Systems and methods for detection of cells using engineered transduction particles

ABSTRACT

Systems and methods for detecting and/or identifying target cells (e.g., bacteria) using engineered transduction particles are described herein. In some embodiments, a method includes mixing a quantity of transduction particles within a sample. The transduction particles are associated with a target cell. The transduction particles are non-replicative, and are engineered to include a nucleic acid molecule formulated to cause the target cell to produce a series of reporter molecules. The sample and the transduction particles are maintained to express the series of the reporter molecules when target cell is present in the sample. A signal associated with a quantity of the reporter molecules is received. In some embodiments, a magnitude of the signal is independent from a quantity of the transduction particle above a predetermined quantity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/388,750, entitled “Systems and Methods for Detection of Cells UsingEngineered Transduction Particles,” filed Dec. 22, 2016, which is adivisional of U.S. patent application Ser. No. 14/480,269, now U.S. Pat.No. 9,456,391, entitled “Systems and Methods for Detection of CellsUsing Engineered Transduction Particles,” filed Sep. 8, 2014, which is acontinuation of U.S. patent application Ser. No. 14/048,974, now U.S.Pat. No. 8,829,473, entitled “Systems and Methods for Detection of CellsUsing Engineered Transduction Particles,” filed Oct. 8, 2013, which is adivisional of U.S. application Ser. No. 13/802,461, now U.S. Pat. No.9,481,903, entitled “Systems and Methods for Detection of Cells UsingEngineered Transduction Particles,” filed Mar. 13, 2013, which claimspriority to and the benefit of U.S. Provisional Application Ser. No.61/779,177, entitled “Non-Replicative Transduction Particles andTransduction Particle-Based Reporter Systems,” filed Mar. 13, 2013, allof which are incorporated herein by reference in their entirety.

BACKGROUND

The embodiments described herein relate to systems and methods fordetection of cells using engineered transduction particles. Moreparticularly, the embodiments described herein relate to methods fordetecting bacteria using replication-deficient transduction particles asa reporter system. The embodiments described herein also relate to acontainer and instrument within which the detection of bacteria can beperformed in an integrated, closed system with walkaway functionality.

Detection of bacteria, especially drug resistant strains, is a criticalstep in diagnosing and limiting spread of bacterial infections. Forexample, MRSA is a drug-resistant version of the common Staphylococcusaureus bacteria that is carried by a significant portion of thepopulation in the U.S. Most infections of MRSA occur in hospitals, andcan have a high mortality rate (MRSA infections kill approximately19,000 people in the U.S. every year). Accordingly, there is a need forefficient, accurate and rapid identification of the bacterial strains(including their phenotype and/or genotype and other molecular targets)that cause infection, such as MRSA. Particularly important is theability to identify the bacterial phenotype and/or genotype and othermolecular targets from a variety of different samples (e.g., humansamples, environmental samples, plant samples, veterinary samples, foodsamples or the like), so that the appropriate treatment and controlregimen can be started in a timely fashion.

One known method for identifying bacteria includes bacterial culture.Culturing is highly sensitive, but often takes two to three days (oreven longer) to yield a result, and is therefore not suitable for rapiddiagnosis or for efficient screening purposes. Known culturing methodsare often performed using systems that require highly trained personnelto perform the assay, and are therefore not suitable for use in avariety of different settings. Known culturing methods are also prone tocontamination, which can result in false positives and/ormisidentification of the bacteria. Moreover, known culturing methodsemploy specifically tailored culture protocols for identification ofvarious bacterial species, thus testing a broad bacteria panel canrapidly elevate the cost.

Direct bacterial immunodetection, that is, detection using an antibodyantigen reaction, is another method for bacterial detection. Knownmethods of immunodetection can produce results more quickly and at alower cost than a culture, but are often limited by the availability ofselective antibodies for the bacterial strain of interest and availableantibodies are prone to cross-reactivity. Such known methods are alsoless sensitive than culturing, so there is often nevertheless arequirement of bacterial amplification that can lengthen the assay time.

Other known methods for detection of bacterial cells include isolationand analysis of nucleic acid such as DNA or RNA. Known methods forisolating nucleic acids from a sample often include several stringentsample preparation steps that require expensive and specializedequipment. In particular, such steps include 1) removing the proteinswithin a sample containing bacteria or cells by adding a protease; 2)breaking down the remaining bulk sample to expose the nucleic acidscontained therein (also referred to as cell lysing); 3) precipitatingthe nucleic acid from the sample; 4) washing and/or otherwise preparingthe nucleic acid for further analysis; 5) analyzing the nucleic acid toidentify the species. After preparing the sample, known analysis methodscan include polymerase chain reaction (PCR), gene sequencing, genefingerprinting, fluorescence, immunoassay, electrochemical immunoassay,microarrays, any other suitable technique or a combination thereof. PCRhas found widespread commercial usage but often requires multiple stepsinvolving expensive reagents and instrumentation. Many known methodsinvolving PCR are not suitable for bench top testing (e.g., they requirerelatively skilled personnel). Moreover, known PCR methods employthermal cycling and/or elevated temperatures, which can increase thecost, time and/or complexity of the analysis. Finally, because PCRmethods for detecting DNA sequences lyse the sample cells, such methodscannot distinguish between live and dead cells.

Some known systems and methods for cell identification include the useof bacteriophages to identify and/or detect certain bacteria. In someknown methods, phages that are tagged with a reporter molecule can beused to target and infect a specific bacterial strain. After infection,the phages can undergo a lytic cycle (i.e., break the cell wall killingthe target bacteria) and/or a lysogenic cycle (i.e., replication of thephage along with the bacteria without killing the bacteria), followed bydetection of the amplified progeny phage. Such known methods relying onphage detection often include limiting or complex steps. For example,some known phage detection-based methods for identification rely onphage replication (during which the bacteria can be lysed), andtypically require cell culturing for facilitating this process. Someknown phage detection-based methods require removal or “unbinding” ofspecifically bound phages from the samples using carefully meteredand/or pH controlled reagents. Moreover, some known phagedetection-based methods rely on careful metering of the amount of phageadded and/or include opening or closing of the reaction chamber toadd/remove reagents, which can lead to contamination and/or prematuremixing of reagents leading to erroneous results and making the assaycomplex in nature.

Other phage-based methods employ bacteriophages that are engineered todeliver into the target bacteria a nucleotide that can include areporter gene, which cause the target bacteria to express a reportermolecule. Some known methods include phages that replicate during theassay, however, which can result in an undesirable lysing of the cellswithin which the reporter molecules are to be produced. Other knownphage-based methods employ bacteriophages in which the replicativefunctions are suppressed during the assay conditions. Such knownmethods, however, are difficult to implement due to the tight range ofconditions (e.g., temperature conditions) under which the replicativefunctions will remain suppressed. Such methods are not easilycontrolled, and thus can result in lytic activity. Still other methodssuggest the use of temperate phages that undergo a lysogenic cycleinstead of a lytic cycle. Such known methods, however, are alsosusceptible to sporadic lytic activity. Incorporation of native phagelife cycles may also lead to limiting of the reporter phage host rangedue to superinfection immunity by target cells that may be lysogenizedwith a prophage. Thus, although known methods of this type have beenperformed in an academic setting, they are not applicable in a clinicalsetting.

In addition to the above-described drawbacks regarding the use ofphage-based methods, known methods do not employ automation orinstrumentation for enabling a “walk away” bacteriophage identificationsystem. For example, many known systems do not accommodate closed systemhandling and/or measurement of a signal that is produced by certainreporter molecules, such as for example, a flash luminescence reaction.Thus, known systems and methods require skilled personnel and intimatehandling of the samples, which can increase the possibility of falsepositives or negatives.

Thus, a need exists for improved apparatus and methods for rapid, costeffective and facile detection and identification of bacterial speciesin clinical samples.

SUMMARY

Systems and methods for detecting and/or identifying target cells (e.g.,bacteria) using engineered viral vectors and/or transduction particlesare described herein. In some embodiments, a method includes mixing aquantity of transduction particles within a sample. The transductionparticles are associated with a target cell. The transduction particlesare non-replicative, and are engineered to include a nucleic acidmolecule formulated to cause the target cell to produce a series ofreporter molecules. The sample and the transduction particles aremaintained to express the series of the reporter molecules when targetcell is present in the sample. A signal associated with a quantity ofthe reporter molecules is received. In some embodiments, a magnitude ofthe signal is independent from a quantity of the transduction particleabove a predetermined quantity.

In some embodiments, a container includes a housing, a delivery member,and an actuator. The housing, which can be removably coupled to areaction chamber, defines a reagent volume. The delivery member iscoupled to the housing and defines a pathway between the reagent volumeand the reaction chamber when the housing is coupled to the reactionchamber. A first end portion of the delivery member is disposed withinthe reagent volume and a second end portion of the delivery member isdisposed outside of the reagent volume. The actuator has a plungerportion disposed within the reagent volume that can be moved within thereagent volume along a longitudinal axis of the housing to produce aflow or reagent from the reagent volume via the pathway. The deliverymember is configured to direct the flow of the reagent exiting thesecond end portion of the delivery member in an exit directionnon-parallel to the longitudinal axis of the housing.

In some embodiments, an instrument includes a retention assembly, anactivation assembly and an actuator. The retention assembly includes afirst gripper, a second gripper and a biasing member. The first gripperand the second gripper are configured to contact a first portion of asample container to limit movement of the sample container. The samplecontainer defines a reaction volume and a reagent volume. The activationmember is movably coupled to the retention assembly, and is configuredto engage a second portion of the sample container to convey a reagentfrom the reagent volume into the reaction volume. The actuator isconfigured to move the activation member relative to the retentionassembly between a first position and a second position. In the firstposition, a surface of the activation member is in contact with asurface of the retention assembly to maintain the first gripper and thesecond gripper in an opened configuration. In the second position, thesurface of the activation member is spaced apart from the surface of theretention assembly such that the biasing member urges the first gripperand the second gripper into a closed configuration. A plunger portion ofthe activation member is configured to move within the reagent volumewhen the activation member moves toward the second position.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of a system for bacteria identification,according to an embodiment.

FIGS. 2 and 3 are schematic illustrations of a cartridge according to anembodiment, in a first configuration and a second configuration.

FIG. 4 illustrates a flow diagram of a method for detecting a targetcell in a sample, according to an embodiment.

FIG. 5 is a schematic illustration of a transduction particle and anengineered nucleic acid molecule contained therein, according to anembodiment.

FIGS. 6A, 6B, and 6C show schematic illustrations of a method foridentification of a target cell, according to an embodiment.

FIG. 7A and 7B show a schematic illustration of transduction particlesinteracting with target cells at a first time (FIG. 7A) and a secondtime (FIG. 7B), according to an embodiment.

FIG. 8 illustrates a flow diagram of a method for the identification ofa viable target cell, according to an embodiment.

FIG. 9 is a schematic illustration of a formulation of an engineerednucleic acid, according to an embodiment.

FIG. 10 illustrates a flow diagram of a method for genotypicidentification of a target cell, according to an embodiment.

FIGS. 11A, 11B, 11C, and 11D show schematic illustrations of genotypicmethod for identification of a target cell, according to an embodiment.

FIGS. 12-14 are schematic cross sectional views of a sample containeraccording to an embodiment, in a first configuration, a secondconfiguration and a third configuration, respectively.

FIGS. 15-17 are side views of a container assembly according to anembodiment, in a first configuration, a second configuration and a thirdconfiguration, respectively.

FIG. 18 is a perspective view of a container assembly according to anembodiment.

FIG. 19 shows an exploded view of the container assembly of FIG. 18.

FIG. 20 shows a top view of a housing included in the container assemblyof FIG. 19.

FIG. 21 shows a perspective bottom view of the housing included in thecontainer assembly of FIG. 19.

FIG. 22 shows a perspective view of a reagent container included in thecontainer assembly of FIG. 19, according to an embodiment.

FIGS. 23-25 are side cross-sectional views of a portion of the containerof FIG. 19 in a first configuration, a second configuration and a thirdconfiguration, respectively.

FIG. 26 shows a perspective view of a container assembly, according toan embodiment.

FIG. 27 shows a perspective bottom view of a housing included in thecontainer assembly of FIG. 26.

FIG. 28 shows a side cross-sectional view of the container assembly ofFIG. 26.

FIG. 29 is a side cross-sectional view of a container assembly,according to an embodiment.

FIGS. 30-32 are side cross-sectional views of a container assemblyaccording to an embodiment, in a first configuration, a secondconfiguration and a third configuration, respectively.

FIG. 33 shows an exploded side cross-section of a container assembly,according to an embodiment.

FIGS. 34-36 are side cross-sectional views of the container assembly ofFIG. 33 in a first configuration, a second configuration and a thirdconfiguration, respectively.

FIGS. 37 and 38 are side cross-sectional schematic illustrations of acontainer assembly according to an embodiment in a first configurationand a second configuration, respectively.

FIGS. 39 and 40 are side cross-sectional schematic illustrations of acontainer assembly according to an embodiment in a first configurationand a second configuration, respectively.

FIG. 41 is side cross-sectional schematic illustration of a containerassembly, according to an embodiment.

FIG. 42 illustrates a flow diagram of a method of signal detection,according to an embodiment.

FIGS. 43-45 are schematic side views of a portion of an instrumentaccording to an embodiment, in a first configuration, a secondconfiguration and a third configuration, respectively.

FIGS. 46-48 are schematic side views of a portion of an instrumentaccording to an embodiment, in a first configuration, a secondconfiguration and a third configuration, respectively.

FIGS. 49 and 50 are schematic side views of a detection portion of aninstrument according to an embodiment, in a first configuration and asecond configuration, respectively.

FIG. 51 shows a perspective view of an instrument, according to anembodiment.

FIG. 52 shows an inclined front view of the instrument of FIG. 51 with alid opened.

FIG. 53 shows an inclines front view of a housing included in theinstrument of FIG. 51.

FIG. 54 shows a back view of the housing shown in FIG. 53.

FIGS. 55-57 show perspective views of a portion of the internalcomponents and subassemblies of the instrument of FIG. 51 with thehousing removed for clarity.

FIG. 58 shows a perspective view of a power supply included in theinstrument of FIG. 51.

FIG. 59 shows a perspective view of a processor included in theinstrument of FIG. 51.

FIG. 60 shows a perspective view of a communications module included inthe instrument of FIG. 51.

FIG. 61 shows a perspective view of the instrument of FIG. 51 in a firstconfiguration.

FIG. 62 shows a perspective view of a cartridge included in theinstrument of FIG. 51.

FIG. 63 shows a perspective view of a cartridge receiver included in theinstrument of FIG. 51.

FIG. 64 is a side view of the cartridge of FIG. 62 and the cartridgereceiver of FIG. 63 in a coupled configuration.

FIG. 65 shows a perspective view of a heater assembly included in theinstrument of FIG. 51.

FIG. 66 is a partially exploded view of the heater assembly of FIG. 65.

FIG. 67 shows a back view of the heater assembly of FIG. 65.

FIG. 68 is a perspective view of a drive assembly included in theinstrument of FIG. 51, according to an embodiment.

FIG. 69 shows a front view of the drive assembly of FIG. 68.

FIGS. 70 and 71 show the drive assembly of FIG. 68 in a firstconfiguration and a second configuration, respectively.

FIG. 72 shows a perspective view of an electronic circuit system forcontrolling the drive assembly included in the instrument of FIG. 51.

FIG. 73 shows a perspective view of a manipulator assembly according toan embodiment included in the instrument of FIG. 51.

FIG. 74 shows an exploded view of the manipulator assembly of FIG. 73.

FIG. 75 shows a side view of the manipulator assembly of FIG. 73 in afirst (or “open grip”) configuration.

FIG. 76 shows a side view of the manipulator assembly of FIG. 73 in asecond (or “closed grip”) configuration.

FIG. 77 shows a perspective view of the manipulator assembly of FIG. 73in second (“closed grip”) configuration and transporting a container.

FIGS. 78 and 79 show side views of the manipulator assembly of FIG. 73in the first (“open grip”) configuration and engaging a container.

FIG. 80 shows a side cross-section of the manipulator assembly of FIG.78 in the open configuration and engaging the container in a “firstplunge” operation.

FIG. 81 shows a side cross-section of the manipulator assembly of FIG.73 in the second (“closed grip”) configuration.

FIGS. 82 and 83 are a side view and a perspective view, respectively, ofthe manipulator assembly of FIG. 73 in a third configuration (“closedgrip”) configuration engaging the container in a “second plunge”operation.

FIG. 84 shows a side cross section of the manipulator assembly of FIG.82.

FIG. 85 shows a perspective view of a detector assembly, according to anembodiment, included in the instrument of FIG. 51.

FIG. 86 shows a top view of a portion of the detector assembly of FIG.85.

FIG. 87 shows a perspective view of the detector assembly of FIG. 85with a housing removed.

FIG. 88 shows an exploded view of the detector assembly of FIG. 85 withthe housing removed.

FIG. 89 shows a perspective view of a shutter included in the detectorassembly of FIG. 85, according to an embodiment.

FIG. 90 shows a side cross-section of the shutter of FIG. 89.

FIGS. 91-94 are side cross-sectional views of the detector assembly ofFIG. 85 in a first configuration, a second configuration, a thirdconfiguration and a fourth configuration, respectively.

FIG. 95 shows a perspective view of a circuitry included in theinstrument of FIG. 51 for controlling the detector assembly of FIG. 85,according to an embodiment.

FIG. 96 illustrates a flow diagram of a method for receiving a signal,according to an embodiment.

FIG. 97 illustrates a flow diagram of a method for manipulating acontainer, according to an embodiment.

DETAILED DESCRIPTION

Systems and methods for detecting and/or identifying target cells (e.g.,bacteria) using engineered viral vectors and/or transduction particlesare described herein. In some embodiments, a method includes mixing aquantity of transduction particles within a sample. The transductionparticles are associated with a target cell. Similarly stated, thetransduction particles are formulated to bind to and deliver a nucleicacid molecule into the target cell. The transduction particles arenon-replicative, and are engineered to include a nucleic acid moleculeformulated to cause the target cell to produce a series of reportermolecules. The sample and the transduction particles are maintained toexpress the series of the reporter molecules when target cell is presentin the sample. A signal associated with a quantity of the reportermolecules is received. In some embodiments, a magnitude of the signal isindependent from a quantity of the transduction particle above apredetermined quantity.

In some embodiments, a method for detecting a target cell includesmixing with a sample a series of transduction particles associated withthe target cell. The transduction particles are engineered to include anucleic acid molecule formulated to cause the target cell to produce aseries of reporter molecules. The transduction particles are devoid of awild-type DNA that is capable of exhibiting wild-type viral functionsassociated with a virus from which the series of transduction particlesare derived. The sample and the series of transduction particles aremaintained such that the series of reporter molecules is only expressedwhen the target cell is present in the sample. A signal associated withthe quantity of reporter molecules is then received. In someembodiments, the magnitude of the signal is independent from thequantity of the series of transduction particles above a predeterminedquantity. Similarly stated, in some embodiments, the strength of thesignal is substantially independent from the quantity of the series oftransduction particles.

In some embodiments, a method for detecting a target cell includesmixing within a sample a series of transduction particles associatedwith the target cell. The series of transduction particles areengineered to be incapable of lysogenic replication and to include anucleic acid molecule formulated to cause the target cell to produce aseries of reporter molecules. The sample and the series of transductionparticles are maintained to express the series of reporter moleculeswhen the sample includes the target cell. The method also includesreceiving a signal associated with a quantity of the series of thereporter molecules.

In some embodiments, a container includes a housing, a first actuatorand a second actuator. The housing is configured to be removably coupledto a reaction chamber (e.g., which can contain a sample including atarget cell). The housing defines a first reagent volume and a secondreagent volume, and includes a delivery portion that defines a firstpathway between the first reagent volume and the reaction chamber and asecond pathway between the second reagent volume and the reactionchamber. The first actuator has a plunger portion disposed within thefirst reagent volume, and an engagement portion configured to bemanipulated to move the plunger portion within the first reagent volume.The second actuator has a plunger portion disposed within the secondreagent volume, and an engagement portion of the second actuator isconfigured to be manipulated to move the plunger portion within thesecond reagent volume. The engagement portion of the second actuator atleast partially surrounds the engagement portion of the first actuator.

In some embodiments, a container includes a housing, a delivery member,and an actuator. The housing, which can be removably coupled to areaction chamber (e.g., that contains a target cell), defines a reagentvolume. The delivery member is coupled to the housing and defines apathway between the reagent volume and the reaction chamber when thehousing is coupled to the reaction chamber. A first end portion of thedelivery member is disposed within the reagent volume and a second endportion of the delivery member is disposed outside of the reagentvolume. The actuator has a plunger portion disposed within the reagentvolume that can be moved within the reagent volume along a longitudinalaxis of the housing to produce a flow or reagent from the reagent volumevia the pathway. The delivery member is configured to direct the flow ofthe reagent exiting the second end portion of the delivery member in anexit direction non-parallel to the longitudinal axis of the housing.

In some embodiments, a container includes a housing, a delivery member,and an actuator. The housing defines a reagent volume and is removablycoupleable to a reaction chamber. The delivery member is coupled to thehousing and defines a pathway between the reagent volume and thereaction chamber when the housing is coupled to the reaction chamber. Afirst end portion of the delivery member is disposed within the reagentvolume and defines a first portion of the pathway. A second end portionof the delivery member is disposed outside the housing and defines asecond portion of the pathway. A center line of the second portion ofthe pathway is angularly offset from a center line of the first portionof the pathway. The actuator has a plunger portion disposed within thereagent volume and is configured to be moved within the reagent chamberalong a longitudinal axis of the housing to produce a flow of a reagentfrom the reagent volume via the pathway.

In some embodiments, a method for detecting a target cell includesplacing a reaction chamber containing a sample and a series of reportermolecules in operable communication with a detector. A reagent isconveyed into the reaction chamber via a delivery member such that thereagent flows along a surface of the reaction chamber and into thesample. In this manner, aeration of the sample and the reagent and/orthe production of bubbles within the sample is minimized The reagent isformulated to react with the series of reporter molecules to enableand/or enhance the production of a signal associated with a quantity ofthe series of reporter molecules. The signal is received by a detector.

In some embodiments, an instrument includes a retention member, anactivation member and an actuator. The retention member is configured tocontact a first portion of a sample container, which defines a reactionvolume and a reagent volume, to limit movement of the sample container.The activation member is coupled to the retention member, and isconfigured to engage a second portion of the sample container to conveya reagent from the reagent volume into the reaction volume. The actuatoris configured to move the activation member relative to the retentionmember between a first position, a second position and a third position.In the first position, the retention member is configured to be spacedapart from the first portion of the sample container. In the secondposition, the activation member is configured to be spaced apart fromthe second portion of the sample container and the retention member isconfigured to contact the first portion of the sample container. In thethird position, the activation member is configured to be engaged withthe second portion of the sample container to convey the reagent suchthat the retention member is in contact with the first portion of thesample container.

In some embodiments, an instrument includes a retention assembly, anactivation assembly and an actuator. The retention assembly includes afirst gripper, a second gripper and a biasing member. The first gripperand the second gripper are configured to contact a first portion of asample container to limit movement of the sample container. The samplecontainer defines a reaction volume and a reagent volume. The activationmember is movably coupled to the retention assembly, and is configuredto engage a second portion of the sample container to convey a reagentfrom the reagent volume into the reaction volume. The actuator isconfigured to move the activation member relative to the retentionassembly between a first position and a second position. In the firstposition, a surface of the activation member is in contact with asurface of the retention assembly to maintain the first gripper and thesecond gripper in an opened configuration. In the second position, thesurface of the activation member is spaced apart from the surface of theretention assembly such that the biasing member urges the first gripperand the second gripper into a closed configuration. A plunger portion ofthe activation member is configured to move within the reagent volumewhen the activation member moves toward the second position.

In some embodiments, an instrument includes a housing and a shutterhaving a portion movably disposed within the housing between a firstshutter position and a second shutter position. The housing defines achannel configured to receive a sample container, and further defines adetection volume configured to place the channel in communication with adetector. The housing including a first seal surface and a second sealsurface. A first portion of the sample container and the first sealsurface are configured to isolate the detection volume from a volumeoutside of the housing when a second portion (e.g., a distal endportion) of the sample container is disposed within the detectionvolume. A seal surface of the shutter and the second seal surface of thehousing are configured to isolate the detection volume from the channelof the housing when shutter is in the first shutter position. Thechannel of the housing is in communication with the detection volumewhen the shutter is in the second shutter position.

In some embodiments, an instrument includes a housing and a shutterhaving a portion movably disposed within the housing between a firstshutter position and a second shutter position. The housing defines achannel configured to receive a sample container, and also defines adetection volume configured to place the channel in communication with adetector. An actuation portion of the shutter is configured to engage adistal end portion of the sample container to move the shutter from thefirst shutter position to the second shutter position when the distalend portion of the container is moved towards the detection volume. Aseal surface of the shutter and a seal surface of the housing areconfigured to isolate the detection volume from the channel of thehousing when shutter is in the first shutter position. The channel ofthe housing is in communication with the detection volume when theshutter is in the second shutter position.

In some embodiments, an instrument includes a housing and a shutterdisposed within the housing between a first shutter position and asecond shutter position. The housing defines a channel configured toreceive a sample container, and also defines a detection volumeconfigured to place the channel in communication with a detector. Theshutter defines a calibration port configured to receive a calibrationlight source, such as, for example, an LED. A seal surface of theshutter and a corresponding seal surface of the housing are configuredto isolate the detection volume from the channel of the housing whenshutter is in the first shutter position. The calibration port is incommunication with the detection volume when shutter is in the firstshutter position. The channel of the housing is in communication withthe detection volume and the calibration port is isolated from thedetection volume when the shutter is in the second shutter position.

In some embodiments, a method for receiving a signal includes receivinga first signal associated with a magnitude of light emission in adetection volume, at a first time. The detection volume is opticallyisolated from a channel by a movable shutter, which is in a firstposition. The method also includes applying a force to a samplecontainer at least partially disposed within a channel such that adistal end portion of the sample container moves the shutter from thefirst position to a second position, and such that the distal endportion of the sample container is disposed within the detection volume.In this configuration, the channel is in optical communication with thedetection volume. The method further includes receiving, a second signalassociated with a magnitude of light emission in the detection volume ata second time, when the distal end portion of the sample container is inthe detection volume.

As described herein, the terms “gene,” “DNA” and “nucleotide” mean thewhole or a portion of the genetic sequence of the target bacteria or thevector.

As described herein, the term “plasmid” means the engineered gene,sequence and/or molecule contained within the vector that includesregulatory elements, nucleic acid sequences homologous to target genes,and various reporter constructs for causing the expression of reportermolecules within a viable cell and/or when an intracellular molecule ispresent within a target cell.

Systems, devices and methods for detecting and identifying target cells(e.g., bacteria) can include a transduction particle that can identifyand bind to the target cell and deliver into the target cell anengineered nucleotide. As shown in the block diagram of FIG. 1, in someembodiments, a system 100 includes a genetically engineered transductionparticle 110, a container 120, a reporter 130, and a detectioninstrument 140. As described in detail herein, the system 100 isconfigured to manipulate, handle and/or actuate the container 120 and/orthe detection instrument 140 such that the transduction particle 110can, when mixed with a sample S that contains a particular target,produce the reporter 130. In this manner, the system 100 and methodsassociated therewith can be thought of as a “switchable” assay, meaningthat no amount of the reporter 130 is present in the sample until theconditions (e.g., the presence of the target cell) are such that thereporter 130 is produced.

The transduction particle 110 can be any suitable particle capable ofdelivering via transduction non-viral DNA and/or RNA into a target cell.For example, in some embodiments, the transduction particle can bederived from a bacteriophage, or can be a non-biologically derivedvector that is capable of introducing nucleic acid molecules into thetarget bacteria in the sample S. The transduction particle 110 isfurther engineered and/or configured to carry an engineered molecule,for example, recombinant DNA, RNA, nucleotide, plasmid, ribozyme,aptamer, and/or protein. In some embodiments, the transduction particle110 does not contain any DNA from the viral vector (e.g., bacteriophage)from which it was derived. Similarly stated, in some embodiments, thetransduction particle is a viral vector devoid of a wild-type DNAcapable of exhibiting wild-type viral functions associated with thevirus from which the viral vector is derived. In some embodiments, atransduction particle includes any of the transduction particlesdescribed herein.

In some embodiments, the transduction particle 110 is incapable ofreplicating via either the lytic or lysogenic cycle. By eliminating allforms of replication from the transduction particle, the target cellswill be maintained (i.e., not destroyed, killed or lysed) during theproduction of the reporter molecules, thereby improving the accuracy andreliability of the methods used therewith. In this manner, the assaysdescribed herein reduce and/or eliminate the likelihood of a falsenegative, making the methods applicable in a clinical setting. Inparticular, because wild-type viral functions of viral particles canexhibit lysogenic replication and require the capability for lyticreplication, attempts to suppress the replicative functions (e.g., thelytic cycle) may not provide sufficient certainty that the lytic cyclewill not result in some population of assays. To demonstrate theadvantages of the use a transduction particle in which the replicationcapability is eliminated, the lytic activity of two temperate S. aureusphages on ten MRSA clinical isolates was examined via plaque assay. Asshown in Table 1, the phage phi11 exhibited lytic activity on each ofthe ten clinical MRSA isolates, and the phage phi80alpha exhibited lyticactivity on six of the ten clinical MRSA isolates. As shown, assaysrelying on the natural lysogenic cycle of phages (e.g., the temperatephages as tested) can be expected to exhibit lytic activitysporadically. Accordingly, in some embodiments, the transductionparticle 110, and other transduction particles described herein, isengineered to be non-replicative or replication deficient (i.e.,incapable of replication).

TABLE 1 PFFGE MRSA Isolate Type phi11 phi80alpha 1. USA200 x 2. USA1000x 3. USA800 x x 4. USA300 x x 5. USA300 x x 6. USA100 x 7. USA300 x x 8.USA100 x 9. USA300 x x 10. USA100 x x

The transduction particle 110 is characterized by being associated withand/or specific to one or more target cells. Similarly stated, thetransduction particle 110 is formulated to bind to and deliver a nucleicacid molecule into the target cell. For example, the transductionparticle can be selected, engineered and/or produced to bind to anybacteria, e.g., Escherichia, Mycobacterium, Staphylococcus, Listeria,Clostridium, Enterococcus, Streptococcus, Helicobacter, Rickettsia,Haemophilus, Xenorhabdus, Acinetobacter, Bordetella, Pseudomonas,Aeromonas, Actinobacillus, Pasteurella, Vibrio, Legionella, Bacillus,Calothrix, Methanococcus, Stenotrophomonas, Chlamydia, Neisseria,Salmonella, Shigella, Campylobacter and Yersinia.

In some embodiments, the non-replicative transduction particle 110, aswell as any of the non-replicative transduction particles describedherein, can be developed by packaging nucleic acid into the structuralcomponents of a virus and/or bacteriophage where the packaged nucleicacid is devoid from exhibiting native viral and/or bacteriophagefunctions that allow for the virus and/or bacteriophage to replicatewhether the replication is via a lytic or lysogenic pathway.

In one embodiment, a plasmid packaging system can be developed in whichthe small terminase gene containing the pac-site of a pac-type prophageis deleted and then complemented via a plasmid. When the lytic cycle ofthe lysogenized prophage is induced, the bacteriophage packaging systempackages plasmid DNA into progeny bacteriophage structural componentsrather than packaging native bacteriophage DNA. The packaging systemthus produces non-replicative transduction particles carrying plasmidDNA.

In another embodiment, genomic island (GI)-packaging systems can beexploited such that exogenous nucleic acid sequences are packaged by thebacteriophage. This can be accomplished by incorporating such exogenousnucleic acids sequences into the GI. Natural GI-packaging systems resultin both non-replicative GI-containing transduction particles as well asnative replicative phage, thus in order to eliminate the native phagefrom this process, the small terminase gene of the prophage is deleted.The small terminase gene sequence contains the pac-site sequence of thenative phage and thus this deletion has the effect of preventing thepackaging of native phage DNA. If at the same time a GI to be packagedincludes its own pac-site and a small terminase gene that expresses asuitable small terminase protein, then only GI DNA will be amenable forpackaging in this system. By incorporating exogenous DNA into thissystem, non-replicative transduction particles that incorporate GI DNAand exogenous DNA can be produced.

The transduction particle 110 can be further produced and/or engineeredto contain genes and/or a nucleic acid molecule for expressing areporter 130 that can be detected (e.g., via the instrument 140). Thereporter 130 can be any one of a bacterial luciferase, a eukaryoticluciferase, a fluorescent protein (e.g., GFP, etc.), an enzyme suitablefor colorimetric detection (e.g., horseradish peroxidase) a proteinsuitable for immunodetection (e.g., protein A, etc.), a peptide orpeptide tag suitable for immunodetection (e.g., 3X FLAG, etc.) and/or anucleic acid that functions as an aptamer or that exhibits enzymaticactivity. More particularly, the transduction particle 110 does notproduce the reporter 130 autonomously and/or does not include thereporter 130. Instead, transduction particle 110 is configured tocommunicate an engineered nucleic acid molecule contained therein intothe target cell, e.g., bacteria, such that the engineered nucleic acidmolecule uses the natural transcription and translation functions of thebacteria DNA to produce the reporter 130. Thus, the reporter 130 can bethought of as a “switchable” reporter, meaning that no amount of thereporter 130 is present in the sample until the conditions (e.g., thepresence of the target cell) are such that the reporter 130 is produced.In this manner, the methods described herein include no washing ofnon-bound reporter 130, no signal subtraction to account for initialquantities of reporter or the like. Thus, the system 100 and the methodsassociated therewith allows for the development of a homogeneous assay.Further, no temperature cycling is required, and heating at a lowtemperature, for example 37 degrees Celsius, for a short time can besufficient.

The reporter system formulated to cause the expression of the reporter130 and any of the reporter systems disclosed herein can be developedfor reporting on the presence of viable bacteria and/or target cells byincorporating into the non-replicative transduction particle 110 (or anyof the other transduction particles disclosed herein) a reportermolecule under the control of a promoter. When this transductionparticle 110 introduces the reporter system into a cell within the hostrange of the transduction particle 110, the promoter is able to drivethe expression of the reporter molecule.

In one embodiment, a MSSA/MRSA reporter assay can be developed and/orperformed using any suitable system and method as described herein (suchas, for example the system 1000). In such embodiments, a non-replicativetransduction particle (e.g., the transduction particle 110, thetransduction particle 160 or the like) is developed from a S.aureus-specific bacteriophage and the bacterial luciferase genes luxABunder the control of a constitutive promoter are incorporated. When thistransduction particle introduces the reporter system into S. aureus, theconstitutive promoter can express luxAB suitable for reporting on thepresence of a viable S. aureus. If in addition, the antibioticcefoxitin, or a similar anti-biotic, is also added prior to orsimultaneously with mixing the transduction particles with S. aureuscells, if the cells do not contain and express the mecA gene, no luxABwill be expressed in the assay, thus indicating that the cells are MSSA(i.e., sensitive to inhibition by cefoxitin). If, however, the cells docontain and express the mecA gene, luxAB will be expressed in the assay,thus indicating that the cells are MRSA (i.e., resistant to inhibitionby cefoxitin).

Although described as being developed for reporting on the presence ofviable bacteria, in other embodiments the reporter 130 and any of theapplicable reporter systems (e.g., the reporter 630) can be developedfor reporting on the presence of target genes within target bacteria. Inthis system a promoter-less reporter gene is placed downstream of anucleic acid sequence that is homologous to a target gene sequence andthis reporter construct is incorporated into a non-replicativetransduction particle. When the transduction particle introduces thereporter construct into a target cell, the reporter gene will not beexpressed unless the target cell contains the target gene and ahomologous recombination event integrates the reporter gene within thetarget gene loci in the target cell such that the reporter gene becomesoperatively linked to the target gene promoter within target cell.

In one such embodiment, a MRSA reporter system can be developed byincorporating into a S. aureus-specific non-replicative transductionparticle (e.g., the transduction particle 110, the transduction particle160 or the like) a reporter construct consisting of a nucleic acidsequence that is homologous to the mecA gene upstream of promoter-lessbacterial luciferase genes, luxAB. When the transduction particleintroduces the reporter construct into a target S. aureus cell, thereporter gene will not be expressed unless the target cell contains thetarget mecA gene and a homologous recombination event integrates theluxAB genes within the mecA gene loci in the target cell such that thereporter gene becomes operatively linked to the mecA gene promoterwithin target cell.

In some embodiments, transduction particle 110, the nucleic acidmolecule contained within the transduction particle 110 and/or thereporter systems associated therewith can include any of the portions ofthe recombinant bacteriophages shown and described in U.S. PatentPublication No. 2010/0112549, entitled “Microorganism Detection Methodand Apparatus,” filed as an International Patent Application on Apr. 18,2008, which is incorporated herein by reference in its entirety.

The sample S can be any sample that possibly contains the targetbacteria, for example, human nasal swab, blood, urine, veterinarysamples, food samples, and/or environmental samples. In someembodiments, the sample S can be a raw sample as obtained from thesource that does not need any preparation, e.g., any separation orwashing steps are not needed. Thus, the system 100 and the methodsassociated therewith are homogeneous. In some embodiments, the sample Scan include a low load of target cell (e.g., nasal swab for MRSAdetection). When used with such samples, the system 100 and the methodsassociated therewith can include a heating and/or incubation period topromote cell replication, which results in higher production of thereporter molecules 130, for example, to generate a signal that isgreater than a minimum signal threshold.

In other embodiments, the sample S can have a higher load of target cell(e.g., positive bacterial blood culture). In such cases, cellreplication is not needed to produce a positive signal sufficient toidentify the target cell. In some such embodiments, the sample can bemaintained at a specific condition e.g., maintained at a temperature ofgreater than or equal to approximately room temperature, 25 degreesCelsius, or 37 degrees Celsius for a predefined time period e.g., lessthan approximately 4 hours. In such embodiments, the temperature andtime period at which the sample S is maintained are such that thequantity of reporter molecules 130 produced is sufficient to generate ameasurable signal, independent of cell replication. In such embodiments,the sample can be maintained at the predefined temperature for a longertime period, e.g., 6 hours, 8 hours, up to 18 hours, or even longer.

In some embodiments, the container 120 that can contain a first reagent,for example, a bacterial nutrient or growth media (e.g., minimalessential media) and/or suitable buffer (e.g. Amies, PBS, TRIS, HEPES,etc) for maintaining the target cell in a viable state, promotingbacterial cell growth or the like. In some embodiments, an antibiotic,for example, cefoxitin can also be included in the first reagent, forexample, when a viable cell assay is intended. A sample S containing thetarget cell can be added to the sample container 120 followed byaddition of the transduction particle 110 to the sample container 120according to any of the methods and using any of the instrumentsdescribed herein. If the target cells are present, the transductionparticle 110 transfers the nucleic acid sequence contained therein intothe target cell such that the nucleotide contained in the transductionparticle 110 is integrated with the genes of the target cell, e.g., hostbacteria. In some embodiments, the container 120 is configured tofluidically isolate the sample S from a region outside the container120. In such embodiments, the transduction particle 110 is maintained influidic isolation from the sample S before the transduction particle 110is mixed therein. In some embodiments, the maintaining can includemaintaining the sample S for a time period such that the quantity of theplurality of the reporter molecules 130 sufficient to produce the signalis produced independent from target cell replication. As describedherein, mixing includes disposing the transduction particle 110 into thesample S while maintaining isolation between the region and thecontainer 120.

In some embodiments, the container 120 can be configured to include anyadditional reagent that is formulated to react with the reportermolecules 130 to produce, catalyze and/or enhance the production of thesignal. For example, the reporter molecule 130 can be luciferase and thecontainer 120 can be configured to contain an aldehyde reagentformulated to trigger, initiate and/or catalyze a luminescence reactionthat can be detected by the production of the signal. In someembodiments, the reagent can include a 6-carbon aldehyde (hexanal), a13-carbon aldehyde (tridecanal) and/or a 14-carbon aldehyde(tetradecanal), inclusive of all the varying carbon chain lengthaldehydes therebetween. In some embodiments, the container 120 can beconfigured to maintain the additional reagent in fluidic isolation fromsample S before being disposed into the sample S. In this manner thetiming of the delivery of the additional reagent into the sample S canbe controlled. In some embodiments, the system 100 can include amechanism for adding the additional reagent at any suitable time and/orin any suitable manner to induce the detectable signal. For example, asdescribed in more detail herein, in some embodiments, the system 100and/or the container 120 can include a mechanism for conveying anadditional reagent into the sample S at a predetermined velocity (orflow rate) to promote the desired level of mixing.

The instrument 140 can be any appropriate instrument to detect thereporter molecule 130 and/or a reaction catalyzed by the reportermolecule 130. For example, the instrument 140 can include optical (e.g.photomultiplier tubes, fluorometers, spectrometers, colorimetricdetection on a lateral flow assay, imaging based detection, CCDs,luminescence detectors for detecting bioluminescence, colorimetric orfluorometric microarrays) and/or electrical detection means (e.g.electrochemical amperometric, potentiometric, conductometric,impedrometric, and/or any other electrochemical sensors).

In some embodiments, the system 100 and/or the methods associatedtherewith can be configured to be a rapid test that does not require anyamplification of the target cells. Using the system 100 and the methodsdescribed herein, a relatively small time, for example, 1 hour, 2 hour,3 hour or 4 hour, up to 18 hours can be needed for the target cellcontaining the nucleic acid sequence from the transduction particle 110to produce a sufficient quantity of reporter molecules 130 that can bedetected. In some embodiments, the system 100 can be configured to be aclosed system after collection of sample S and/or addition oftransduction particle 110. Said another way, in some embodiments, thecontainer is maintained in fluidic isolation from the externalenvironment after the addition of the sample S. This can, for example,reduce chances of contamination. As described above, because the system100 can accommodate raw sample, the system 100 and the methodsassociated therewith do not require any washing or fluid transfer stepsaway from the sample S. The system 100 can therefore be easy to operate,be rapid, inexpensive, and be easily automated. In some embodiments, thesystem 100 can be a platform system that can be configured to operate invarious regimes, for example, viable cell reporting, gene reporting,measuring bacterial resistance and/or susceptibility to antibiotics,and/or bacterial toxin detection, etc. Additional examples of componentsand methods associated with and/or complementary to the system 100 aredescribed further.

FIGS. 2 and 3 are schematic illustrations of a system 1000 according toan embodiment. The system 1000 is configured to be communicativelycoupled to any suitable laboratory information system (LIS) 1900, andincludes an instrument 1100 that is configured to manipulate and/orreceive a container 1700. The system 1000 can be used to identify targetcells in a clinical environment according to any suitable method, suchas any of the methods described herein.

The container 1700 can be any suitable container that can be manipulatedand/or actuated by the instrument 1100 or any other instrumentsdescribed herein. The container 1700 defines an internal volume withinwhich a sample S can be disposed as shown by arrow AA. In someembodiments, the container 1700 can include a solution 1702 disposed inthe internal volume of the container 1700 for interacting with thesample S. The solution 1702 can be predisposed in the internal volumedefined by the container 1700 or added after the sample S is conveyedinto the container 1700. The solution 1702 can include, for example, abacterial nutrient and/or growth media (e.g., undefined medium, definedmedium, differential medium, minimal media, selective media, etc.) toenable bacteria to grow and multiply, a buffer to maintain pH (e.g.,Amies, PBS, HEPES, TRIS, TAPSO, Bicine, MES, MOPS, Tricine, PIPES, SSC,succinic acid, etc.) and/or a surfactant (e.g., Tween 20, Tween 80,TritonX, X-114, CHAPS, DOC, NP-40 CTAB, SDS, etc.). In some embodiments,the solution 1702 can also include antibiotics (e.g. cefoxitin,oxacillin, cefotetan, amoxycillin, penicillin, erythromycin,azythromycin, cephalosporins, carbapenems, aminoglycosides,sulfonamides, quinolones, oxazolidinones, etc.). The inclusion ofantibiotics can kill or otherwise prevent the expression and/orgeneration of a signal from reporter molecule from all drug-susceptiblebacteria, e.g., in a bacteria cell viability and/or susceptibility assayof the types shown and described herein.

In some embodiments, the solution 1702 can be tailored to enhancegrowth, shorten lag phase, sustain, and/or attack a particular targetcell, e.g., bacterium. In some embodiments, specific versions of thesolution 1702 can be employed for specific target cells and/or samples.For example, a first preparation of the solution 1702 can be tailoredfor nasal swab samples containing MRSA, a second preparation of thesolution 1702 can be tailored for urine samples containing E. coli, athird preparation of the solution 1702 can be tailored for stool samplescontaining C. difficile, and the like

The container 1700 can be configured to receive a first reagent 1710containing a transduction particle and/or an engineered viral vector asshown by the arrow BB (FIG. 2). In some embodiments, the container 1700can further receive any other reagents in connection with theimplementation of any of the methods described herein. In someembodiments, the transduction particle 1710, and/or any other reagentscan be predisposed in the container 1700, such that, for the example thevector 1710 or any other reagents do not need to be separately added tothe container 1700 after the sample S is placed therein. For example, insome embodiments, the transduction particle 1710 can be disposed in acap or separate portion (not shown) of the container 1700 such that therespective solutions (e.g., the solution 1702, the sample S and thetransduction particle 1710) can remain isolated from each other duringshipping, initial handling or the like. The container 1700 can befurther configured to convey the respective solutions into the interiorvolume of the container 1700 at a suitable time. For example, in someembodiments, the cap can have frangible portions that can be broken bythe instrument 1100 and/or the user at a desired time. For example, thefrangible portions can be broken using plungers, crushing manually, orany other suitable mechanism. In some embodiments, the container 1700can be multi-portion container such that, for example, the container1700 can have frangible portions, each portion containing a separatedfluid. The container 1700 can be configured such that while fluids canbe predisposed in the container 1700 and urged to mix at specific times,the container 1700 does not contain any fluid transfer paths, fluidtransfer mechanisms (e.g., electrophoretic transfer, electrokinetictransfer, pumps, etc.), valves and/or any complex fluid transportschemes.

The container 1700 can be any suitable container for containing thesample S in a manner that permits the monitoring, identification and/ordetection of a target cell, e.g., bacteria, within the sample S. In someembodiments, at least a portion of the container 1700 can besubstantially transparent, for example, to allow viewing, and/or opticalmonitoring of the contents contained therein. The container 1700 can beany suitable size or shape, for example, cylindrical square,rectangular, elliptical, conical, etc. The container 1700 can beconstructed from any suitable material, for example, glass, plastic,acrylic, etc. In some embodiments, the container 1700 can be acommercially available container, for example a centrifuge tube, aneppendorf® tube, a glass vial, flat bottomed vial/tube, round bottomedvial/tube or any other suitable container. In some embodiments, thecontainer 1700 can also include additional components, for example,swabs for collecting patients' samples, cap to protect container 1700from atmosphere and/or containing assay reagents, labels foridentification, bar codes, RFID tags, etc.

Sample S and any other samples described herein can be any suitablesample S that can potentially contain the target cell, e.g., bacteria.For example, the sample S can be a human sample (e.g., a nasal swab,mucosal swab, saliva sample, blood sample, urine sample, fecal sample,tissue biopsy, bone marrow and/or cerebrospinal fluid), veterinarysample, food sample, plant sample, and/or environmental sample. In someembodiments, the sample S can be a raw and substantially unprocessedsample. In such embodiments, the system 1000 (including the container1700 and/or the instrument 1100) is configured such that nomodifications to the sample are required to run the methods associateddescribed in connection with the system 1000. In other embodiments,however, the sample S can undergo minor processing, for example,filtration, sedimentation or any other process required to produce asuitable sample. Such processing can be performed by any suitablemechanism of the instrument 1100.

The transduction particle and/or engineered viral vector 1710 and any ofthe transduction particles and/or vectors disclosed herein, can be anysuitable transduction particle that can specifically identify and bindto a target cell, and perform the functions described herein. In someembodiments, the transduction particle 1710 can be derived from abacteriophage. Examples of suitable transduction particles 1710 caninclude vectors biologically derived from, for example, T2, T4, T7, T12,R17, M13, MS2, G4, p1, enterobacteria phage P4, Phi X 174, N4,pseudomonas phage, lambda phage, and/or any other vector. In someembodiments, the transduction particle 1710 includes modified DNA fromthe phage from which the vector 1710 is derived. In some embodiments,the biologically derived transduction particle 1710 does not include anyDNA associated with the phage from which it was derived. Said anotherway, the transduction particle 1710, e.g., a vector, is devoid of awild-type DNA capable of exhibiting wild-type viral functions associatedwith a virus from which the transduction particle 1710 is derived. Insome embodiments, the lack of any phage DNA removes the capability ofthe transduction particle 1710 to reproduce, replicate or propagate.Said another away, after infecting the bacteria, neither the lytic cyclenor the lysogenic cycle of the transduction particle 1710 and/or thetarget bacteria can cause the transduction particle 1710 to multiply oramplify. Similarly stated, in some embodiments, the transductionparticle and/or engineered viral vector 1710 is non-replicative, i.e.,cannot undergo lytic or lysogenic replication.

In some embodiments, the transduction particle and/or engineered viralvector 1710 can be formulated, selected and/or engineered to includeand/or carry an engineered molecule, for example, recombinant DNA, RNA,nucleic acid sequence, nucleotide, plasmid, ribozyme, aptamer and/orprotein. In some embodiments, the transduction particle 1710 can beconfigured to specifically identify and detect the presence of a viabletarget bacteria e.g., Escherichia, Mycobacterium, Staphylococcus,Listeria, Clostridium, Enterococcus, Streptococcus, Helicobacter,Rickettsia, Haemophilus, Xenorhabdus, Acinetobacter, Bordetella,Pseudomonas, Aeromonas, Actinobacillus, Pasteurella, Vibrio, Legionella,Bacillus, Calothrix, Methanococcus, Stenotrophomonas, Chlamydia,Neisseria, Salmonella, Shigella, Campylobacterand Yersinia. In someembodiments, the transduction particle 1710 can be configured tospecifically identify a bacteria genotype including specific genetargets and other molecular targets indicative of the genotype and/orphenotype of the bacteria, e.g., methicillin resistant Staphylococcusaureus (MRSA), E. coli, Salmonella, C. difficile, vancomycin-resistantEnterococci (VRE), or any other bacteria. In one such embodiment, theplasmid can include a nucleic acid molecule that is homologous for aspecific gene sequence associated with the DNA of the target bacteria.For example, the transduction particle 1710 can be engineered and/orconfigured to include a plasmid that incorporates nucleic acid sequenceshomologous to the mecA gene found in MRSA, e.g., in an assay for thedetection of MRSA (as described above).

The transduction particle 1710 can be further configured to containgenes and/or a nucleic acid molecule for expressing a detectablereporter molecule 1730. The reporter molecule 1730 can be any one of abacterial luciferase, eukaryotic luciferase, fluorescent protein, enzymesuitable for colorimetric detection, protein suitable forimmunodetection, peptide suitable for immunodetection or a nucleic acidthat functions as an aptamer or that exhibits enzymatic activity. Insome embodiments, a reagent (or substrate, not shown in FIGS. 2 and 3)can be added to the solution 1702 to urge the reporter molecule 1730 toproduce a detectable signal. For example, in some embodiments,tridecanal can be added to allow the luciferase to catalyze aluminescence reaction that can be detected. In some embodiments, two ormore transduction particles 1710 specific towards two separate targetcells can be used together in the same reagent, for example, to detectmultiple bacteria simultaneously.

The instrument 1100 includes the detector 1200, and is configured toreceive, manipulate and/or handle the container 1700 to convey, mixand/or add the sample S, the solution 1702 and/or the transductionparticles 1710, and detect and/or identify a constituent within thesample S. In particular, the instrument 1100 can include any suitablesystems/mechanisms (not shown in FIGS. 2 and 3) forhandling/manipulating the container 1700. For example, the instrument1100 can include receptacles, racks, vices, jaws, grippers or any othersuitable mechanism for removably receiving the container 1700. In someembodiments, the instrument 1100 can include mechanisms for manipulatingand/or changing the configuration of the container 1700. For example,the instrument 1100 can include plungers, conveyor belts, stepper motors(e.g. to move and position the container 1700 in X/Y/Z plane), rollers,shakers, clamps, X/Y movable tables, encoders, any other instrumentationfor positioning or manipulating the container 1700 or a combinationthereof. For example, the container 1700 can be disposed in a receptacleincluded in the instrument 1100 that can transport the container 1700via a conveyor belt to a location in the instrument 1100 (see e.g., FIG.3) where the detector 1200 can interface with the container 1700 anddetect the signal produced by the reporter molecule 1730 that indicatesthe presence of target cell (e.g., bacteria). In some embodiments, theinstrument 1100 can include a gripper and an actuator mechanism,configured such that the gripper prevents and/or limits movement of thecontainer 1700 when signal is being detected by the detector 1200. Inthis manner, the instrument 1100 can minimize signal noise by holdingand maintaining the container 1700 at a predetermined distance from thedetector 1200. Moreover, such a mechanism can ensure that the positionof the container 1700 is maintained when the actuator actuates thecontainer 1700, for example, to convey fluid from one portion of thecontainer 1700 to another.

In some embodiments, the container 1700 and/or the instrument 1100 canalso include light seal mechanisms, for example, shutters, to light sealthe container 1700. In this manner, the instrument can limit and/orprevent ambient light from interfering with the signal produced by thereporter 1730. Such systems can also limit and/or prevent any undesiredmotion of the container 1700 during signal detection. In someembodiments, the container 1700 and instrument 1100 are configured suchthat the entire process including loading of the container 1700,handling/manipulation of the container 1700 by the instrument 1100, andsignal detection by the detector 1200 occurs in a closed process. Saidanother way, detection of bacteria in the container 1700 by theinstrument 1100 can be performed without opening the container 1700,does not require fluid handlers or any reagents in the instrument, anddoes not require any sample manipulation.

Although only one container 1700 is shown in FIG. 2 and FIG. 3, in otherembodiments the instrument 1100 can be configured to receive a series ofcontainers 1700. For example, the instrument 1100 can include acontainer rack or magazine, within which a user can removably disposemultiple containers 1700 that can contain multiple samples S foranalysis. In some embodiments, the containers 1700 can be loaded on theinstrument 1100 in a batch process. In other embodiments, the containers1700 can be delivered to the instrument 1100 in a “flow through”process. For example, the containers 1700 can be disposed on a conveyorbelt that can deliver a plurality of containers 1700 sequentially to thereader of the instrument 1100. In some embodiments, the instrument 1100can be automated and be configured for “walk away” analysis. Forexample, the user can load a plurality of containers 1700 for analysis,on the instrument 1100 and walk away. The instrument 1100 canautomatically perform container 1700 manipulation and detection on allthe containers 1700.

The detector 1200 can be any suitable detector than can detect thesignal produced by the reporter molecule 1730. For example, the detector1200 can be an optical detector such as, for example, a fluorescencedetector (e.g. to detect a fluorescent reporter molecule such as GFP,etc.), a luminescence detector (e.g. to detect bioluminescence producedby a reporter molecule such as luciferase), color detector (e.g. todetect a colored precipitant produced by a reporter enzyme such as HRP),a spectrometer, and/or an image capture device. In some embodiments, thedetector 1200 can further include a light source associated with themechanism for detection. Although described as being primarily based onoptical detection, in some embodiments, the detector 1200 can be anelectrochemical detector. For example, the detector 1200 can include anamperometric detector, potentiometric detector, conductometric, and/orimpedometric detector, configured to detect a current, voltage, orconductance, resistance/impedance change produced by the reportermolecules 1730. In some embodiments employing electrochemical detection,the detector 1200 can be configured to come in physical contact with thesample solution 1706 (FIG. 3) that contains the sample S, solution 1702,transduction particle 1710, reporter molecule 1730, and/or any othersubstrate that can be necessary for inducing a signal from the reportermolecule.

In some embodiments, the detector 1200 can use other detection methods,e.g., surface acoustic wave, surface plasmon resonance, Ramanspectroscopy, magnetic sensors, and/or any other suitable detectionmethod known in the art. In some embodiments, the detector 1200 can onlyprovide a qualitative answer, for example, a YES/NO answer on thepresence of target cell. In other embodiments, however, the detector1200 can quantify the target cell, for example, determine the cfu/ml oftarget bacteria in the sample S according to any of the methodsdescribed herein. In some embodiments, the detector 1200 can include anend read system, .e.g., to allow flexible placement of a label on thecontainer 1700. In some embodiments, the end read system includes directcontact of a transparent end of the container 1700 with the detector,e.g., to minimize optical instruments and/or background signalinterference. In some embodiments, the detector 1200 is devoid of anincident light source. Said another way, no external light is needed forsignal detection from the reporter molecules 1730 produced by the targetcell disposed in the container 1700.

In some embodiments, the systems 100,1000, or any other systemsdescribed herein can be used to identify and/or detect a target cell,such as a bacteria. In particular, in some embodiments, the system 1000can be used in conjunction with a replication-deficient transductionparticle to identify and/or detect a target cell. By employing areplication-deficient transduction particle, the likelihood of a falsenegative (e.g., caused by cell destruction from the lytic cycle) isminimized and/or eliminated, thereby producing a result that is suitablein a clinical setting. Such methods can be used, for example, as ascreening tool in hospitals. In particular, FIG. 4 is a flow chart of amethod 150 according to an embodiment.

As shown in FIG. 4, the method 150 includes mixing with a sample asubstance including transduction particles associated with a targetcell, 152. Similarly stated, the transduction particles are formulatedto bind to and deliver a nucleic acid molecule into the target cell. Thetransduction particles are engineered to include a nucleic acid moleculeformulated to cause the target cell to produce a series of reportermolecules. The series of transduction particles are non-replicative.Similarly stated, the transduction particles can be formulated and/orengineered to be incapable of lysogenic or lytic replication. In thismanner, the target cells will be maintained (i.e., not destroyed, killedor lysed) during the production of the reporter molecules. Thus, themethod 150 reduces and/or eliminates the likelihood of a false negative,which can result when the target cells are lysed thereby preventingand/or reducing the production of the reporter molecules.

The transduction particles can be any suitable transduction particles ofthe types shown and described herein. For example, in some embodiments,the transduction particles can be an engineered viral vector devoid of awild-type DNA capable of exhibiting wild-type viral functions associatedwith a virus from which the viral vector is derived. In someembodiments, the transduction particles can be engineered to be derivedfrom a bacteriophage. For example, in some embodiments, the transductionparticles can be developed by packaging nucleic acid into the structuralcomponents of a virus and/or bacteriophage where the packaged nucleicacid is devoid from exhibiting native viral and/or bacteriophagefunctions that allow for the virus and/or bacteriophage to replicatewhether the replication is via a lytic or lysogenic pathway.

In one embodiment, a plasmid packaging system can be developed in whichthe small terminase gene containing the pac-site of a pac-type prophageis deleted and then complemented via a plasmid. When the lytic cycle ofthe lysogenized prophage is induced, the bacteriophage packaging systempackages plasmid DNA into progeny bacteriophage structural componentsrather than packaging native bacteriophage DNA. The packaging systemthus produces non-replicative transduction particles carrying plasmidDNA.

In another embodiment, genomic island (GI)-packaging systems can beexploited such that exogenous nucleic acid sequences are packaged by thebacteriophage. This can be accomplished by incorporating such exogenousnucleic acids sequences into the GI. Natural GI-packaging systems resultin both non-replicative GI-containing transduction particles as well asnative replicative phage, thus in order to eliminate the native phagefrom this process, the small terminase gene of the prophage is deleted.The small terminase gene sequence contains the pac-site sequence of thenative phage and thus this deletion has the effect of preventing thepackaging of native phage DNA. If at the same time a GI to be packagedincludes its own pac-site and a small terminase gene that expresses asuitable small terminase protein, then only GI DNA will be amenable forpackaging in this system. By incorporating exogenous DNA into thissystem, non-replicative transduction particles that incorporate GI DNAand exogenous DNA can be produced.

In some embodiments, the transduction particles can be selected,engineered and/or formulated to specifically bind to, and transfer thenucleic acid molecule contained therein into the viable cells. Forexample, the transduction particles can be selected, engineered and/orproduced to bind to and deliver a nucleic acid molecule into anybacteria, e.g., Escherichia, Mycobacterium, Staphylococcus, Listeria,Clostridium, Enterococcus, Streptococcus, Helicobacter, Rickettsia,Haemophilus, Xenorhabdus, Acinetobacter, Bordetella, Pseudomonas,Aeromonas, Actinobacillus, Pasteurella, Vibrio, Legionella, Bacillus,Calothrix, Methanococcus, Stenotrophomonas, Chlamydia, Neisseria,Salmonella, Shigella, Campylobacter and Yersinia.

The sample and the series of transduction particles are maintained suchthat the series of the reporter molecules is produced when the sampleincludes the target cell, 154. Similarly stated, the sample and theseries of transduction particles are maintained to express the pluralityof reporter molecules when the target cell is present in the sample. Inthis manner, production of the reporter molecules, and the detectionthereof, indicates that the target cell is present in the sample. Moreparticularly, the transduction particles do not produce the reportermolecules autonomously and/or do not include the reporter molecule.Instead, transduction particles are engineered, configured and/orformulated to communicate the nucleic acid molecule contained therein(i.e., an engineered plasmid) into the target cell. Upon being deliveredinto the target cell, reporter molecules are produced using the naturaltranscription and translation functions of the target cell and via theexpression of a reporter gene that is operatively linked to a promoterthat is included in the nucleic acid molecule. Thus, the method 150employs a “switchable” reporter, meaning that no amount of the reportermolecules is present in the sample until the conditions (e.g., thepresence of the target cell) are such that the reporter molecules areproduced. Notably, because the method 150 employs a “switchable”reporter molecule, no washing and/or removal of the transductionparticles and/or other constituents within the sample is necessary. Thereporter molecule can be any one of a bacterial luciferase, eukaryoticluciferase, fluorescent protein, enzyme suitable for colorimetricdetection, protein suitable for immunodetection, peptide suitable forimmunodetection or a nucleic acid that functions as an aptamer or thatexhibits enzymatic activity.

In some embodiments, the method 150 can be used as a viable cellreporter assay. When the method 150 is used as a viable cell reporterassay, in some embodiments, antibiotics (e.g., cefoxitin) can be addedto and/or mixed with the sample to kill and/or eliminate alldrug-susceptible target cells (e.g., in a bacteria) thereby allowing themethod 150 to be used to identify particular target cell drug resistantphenotype (e.g., methicillin resistant Staphylococcus aureus (MRSA),Salmonellavancomycin-resistant Enterococci (VRE)) within the sample.

For example, in some embodiments, a MSSA/MRSA reporter assay can bedeveloped in accordance with the method 150. In such embodiments, anon-replicative transduction particle (e.g., the transduction particle110, the transduction particle 160 or the like) is developed from a S.aureus-specific bacteriophage and the bacterial luciferase genes luxABunder the control of a constitutive promoter are incorporated. When thistransduction particle is mixed with the sample (operation 152) thusintroducing the reporter system into S. aureus, the constitutivepromoter can express luxAB suitable for reporting on the presence of aviable S. aureus, as discussed below with reference to operations 154and 158. If in addition, the antibiotic cefoxitin is also added prior toor simultaneously with mixing the transduction particles with S. aureuscells, if the cells do not contain and express the mecA gene, no luxABwill be expressed in the assay, thus indicating that the cells are MSSA.If, however, the cells do contain and express the mecA gene, luxAB willbe expressed in the assay, thus indicating that the cells are MRSA.

In other embodiments, the nucleic acid molecule can be formulated tocause the target cell to produce the series of reporter molecules onlywhen the target cell includes a particular target gene (e.g., a drugresistant gene, a drug-susceptibility gene, a toxin, or a speciesspecific gene). For example, in some embodiments, the method 150 can beperformed in conjunction with a transduction particle and/or reportersystem in which a promoter-less reporter gene is placed downstream of anucleic acid sequence that is homologous to a target gene sequence andthis reporter construct is incorporated into a non-replicativetransduction particle. When the transduction particle introduces thereporter construct into a target cell, the reporter gene will not beexpressed unless the target cell contains the target gene and ahomologous recombination event integrates the reporter gene within thetarget gene loci in the target cell such that the reporter gene becomesoperatively linked to the target gene promoter within target cellallowing for the expression of the reporter genes driven by the targetgene promoter.

In one such embodiment, a MRSA reporter system can be developed byincorporating into a S. aureus-specific non-replicative transductionparticle (e.g., the transduction particle 110, the transduction particle160 or the like) a reporter construct consisting of a nucleic acidsequence that is homologous to the mecA gene upstream of promoter-lessbacterial luciferase genes, luxAB. When the transduction particleintroduces the reporter construct into a target S. aureus cell, thereporter gene will not be expressed unless the target cell contains thetarget mecA gene and a homologous recombination event integrates theluxAB genes within the mecA gene loci in the target cell such that thereporter gene becomes operatively linked to the mecA gene promoterwithin target cell allowing for the expression of luxAB driven by themecA gene promoter.

The sample with the transduction particles mixed therein can bemaintained at any suitable temperature and for any suitable time topromote production of the reporter molecules and/or growth of the targetcells within the sample. For example, in some embodiments the sample andthe transduction particles are maintained at a temperature of greaterthan or equal to room temperature, 25 degrees Celsius, or 37 degreesCelsius, and for a predefined time period of less than 2 hours, 2 hours,3 hours, 4 hours, 6 hours, up to 18 hours or even more, inclusive of anyranges therebetween. In this manner, the maintaining of the sample atthe predefined temperature for the predefined time period is sufficientto generate a quantity of the series of reporter molecules sufficient toproduce a measurable signal. In some embodiments, the maintaining needonly be sufficient to promote the production of the reporter moleculesin the target cells initially present in the sample. Said another way,in some embodiments, the conditions under which the sample is maintained(e.g., the temperature and/or duration) prior to the detection operationneed not be sufficient to promote repeatable target cell replication.

In some embodiments, the method optionally includes disposing a secondsubstance into the sample, 156. The second substance can be formulatedto react with the series of reporter molecules to catalyze, enhance theproduction of and/or produce a measureable signal, such as, for example,a luminescence signal, a fluorescence signal, a color-based signal, achemical signal or electrochemical signal. For example, in someembodiments, the nucleic acid molecule can include luxA/luxB sequencessuch that the reporter molecule produced can be luciferase. In suchembodiments, the second substance (or reagent) can be an aldehydereagent (e.g., tridecanal). The tridecanal urges the luciferase toproduce luminescence that can be measured. In some embodiments, thereagent can include a 6-carbon aldehyde (hexanal), a 13-carbon aldehyde(tridecanal) and/or a 14-carbon aldehyde (tetradecanal), inclusive ofall the varying carbon chain length aldehydes therebetween. In someembodiments, the reagent formulation can also include a maintainingmedium and/or buffer, e.g., TSB broth, citrate buffer, etc., asurfactant, e.g., Tween 20, and be adjusted to a predetermined pH, e.g.,pH 3.

The identification of the target cell is performed by receiving a signalassociated with a quantity of the series of the reporter molecules, 158.The signal can be measured using any suitable detector as describedherein, (e.g., the detector 1200 of the instrument 1100 described above,the detector 11200 of the instrument 11000 described below, or thelike). In some embodiments, the magnitude of the signal is independentfrom a quantity of transduction particles mixed with the sample. Moreparticularly, because the transduction particles are not “tagged” withthe reporter molecule (i.e., they do not include the reporter molecule),the strength of the signal is dependent not upon the initial quantity ofthe transduction reporters, but rather upon the production of thereporter molecules by the target cell. Thus, the signal is independentfrom the quantity of transduction particle (when the quantity is abovesome de minimus amount or lower threshold).

Any of the steps of the method 150 can be performed using any of thecontainers and/or instruments described herein. For example, the method150 can be performed using the containers 1700, 2700, 3700, 4700, etc.and the instrument 1100, 11000 or the like. For example, in someembodiments, the sample can be disposed within a portion of a containerthat is fluidically isolated from a region outside the container. Forexample, the sample can be disposed or maintained in a reaction chamber(e.g., the reaction chamber 3732 or 4732 of the container assemblies3700 and 4700, respectively) that is sealed and/or closed via a reagentmodule (e.g., the reagent module 3740 or 4740). In some embodiments, thetransduction particles can also be maintained in fluidic isolation fromthe sample before the mixing, e.g., in an internal volume of thecontainer assembly (e.g., such as the reagent module 3740 or 4740). Insome embodiments, the mixing includes disposing the transductionparticles into the sample while maintaining fluidic isolation betweenthe container and an outside region. In this manner, the method can beperformed in a closed system and/or a homogeneous assay. In someembodiments, the reagent can also be maintained in fluidic isolationfrom the sample before disposing, e.g., stored in an internal volume ofthe container. In some embodiments, the reagent can also be disposedinto the sample while maintaining fluidic isolation between thecontainer and the outside region.

In some embodiments, the system 1000 and/or the method 150 (or any othersystems and methods described herein) can be used to identify and/ordetect a drug resistant strain of bacteria. In some embodiments, amethod according to an embodiment can use bacterial viability as themethod of identification, using viral vectors and/or transductionparticles associated with a particular target bacteria. Moreparticularly, in some embodiments, the transduction particles can be abiologically derived viral vector engineered, selected and/or formulatedto perform a detection function. For example, as shown in FIG. 5, insome embodiments, a transduction particle 160 can be derived from abacteriophage in accordance with any of the methods and descriptionsherein. Similarly stated, the transduction particle 160 can include thestructural proteins and/or envelope from a bacteriophage. In particular,the transduction particle 160 can be engineered to include a capsid 162of a phage engineered to retain the capability to bind to the targetbacteria (e.g., via the sheath 164, tail fibers 166 and/or base plate168. In this manner, the transduction particle 160 can selectivelyidentify, bind to and deliver a nucleic acid molecule 170 into a desiredtarget bacteria.

As shown in FIG. 5, the transduction particle 160 contains a nucleicacid molecule 170 formulated to cause the target cell to produce aplurality of reporter molecules. In some embodiments, the transductionparticle 160 and/or the nucleic acid molecule 170 are substantiallydevoid of the native DNA of the phage from which the transductionparticle 160 is derived. Similarly stated, the DNA of the phage fromwhich the transduction particle 160 is replaced by the engineeredplasmid or nucleic acid molecule 170. Said another way, the transductionparticle 160 is substantially devoid of a wild-type DNA capable ofexhibiting wild-type viral functions, such as replication functions,associated with a phage from which the transduction particle 160 isderived. Thus, in such embodiments, the transduction particle 160 is“replication-deficient” or incapable of reproducing or replicating byany means (e.g., by lysogenic and/or lytic replication).

As shown in FIG. 5, the engineered nucleic acid molecule 170 in thetransduction particle 160 contains a reporter sequence 174 that providesinstructions for the target bacteria to express a reporter molecule. Forexample, the reporter sequence 174 can include promoter less sequencesluxA/luxB for expressing luciferase, which cannot be expressed by thetransduction particle 160 autonomously. Rather after the luxA/luxBsequences are inserted into the target bacteria by the transductionparticle 160 and are operatively coupled with the target bacteria DNA,the natural transcription machinery of the target bacteria is used toexpress the reporter molecule, i.e., luciferase. In other embodiments,the reporter sequence 174 can also include a promoter included with theluxA/luxB sequences. In such embodiments, after the promoter coupledluxA/luxB sequences are inserted into the target bacteria by thetransduction particle 160, the promoter coupled luxA/luxB sequences canexpress luciferase without coupling with the target bacteria DNA.

As described herein, in some embodiments, the transduction particle 160can be engineered for reporting on the presence of viable bacteria. Insuch embodiments, the transduction particle 160 is engineered toidentify and/or recognize a target bacteria, attach thereto andcommunicate the engineered nucleic acid 170 into the target bacteria.Upon being conveyed into the target bacteria, the engineered nucleicacid 170 can produce reporter molecules due to the incorporation of apromoter operatively linked to the reporter genes (e.g., the reportersequence luxA/luxB as described before herein) within the engineerednucleic acid. The engineered nucleic acid 170 then causes the targetbacteria to produce the reporter molecules using the naturaltranscription and translation systems of the target bacteria. In suchviable cell reporter embodiments, further specificity can be achieved byeliminating all other phenotypes. For example, in some embodiments, MRSAcan be identified in a cell viability assay by killing or otherwisesuppressing signal generation from all non-drug resistant phenotypeswithin the sample by using antibiotics.

As shown schematically in FIGS. 6A, 6B, and 6C, the transductionparticle 160 can be used to detect target bacteria 180 in any sample S.In the first operation (indicated by the schematic illustration 192),the transduction particle 160 is brought into contact and/or mixed withthe sample S containing the target bacteria 180. The transductionparticle 160 can be mixed with and/or introduced into the sample S usingany suitable methods or mechanisms as described herein. The transductionparticle 160 is selected, formulated and/or engineered to specificallyidentify and bind to the cell wall of the target bacteria 180, as shownby arrow A. At operation 194, the transduction particle 160 communicatesthe engineered nucleic acid 170 contained therein, into the targetbacteria 180 cytoplasm, as shown by arrow B. In some embodiments, whenviable cell reporting is performed, the engineered nucleic acid 170 caninclude the reporter sequence 174 (e.g., luxA/luxB) coupled to apromoter and configured to cause the production of reporter molecules inthe target cell 180, as shown in operation 196.

The presence of reporter molecules 130 indicates that the targetbacteria 180 are present in the sample S. Accordingly, the reportermolecules 130 are “switchable” (i.e., only present under certainconditions). Moreover, because in some embodiments the transductionparticle 160 is non-replicative (i.e., incapable of lytic or lysogenicreplication), the target bacteria 180 remain alive or otherwiseun-inhibited during the assay. Therefore, the systems and methodsdescribed herein can be used to detect live bacteria. Furthermore, whilenot shown in FIGS. 6A, 6B, or 6C, an antibiotic (e.g., cefoxitin) can beadded to the sample S, for example, before adding the transductionparticle 160. The antibiotic can eliminate or otherwise inhibit allnon-antibiotic resistant bacteria (e.g., MSSA) such that only theantibiotic resistant strains (e.g., MRSA) remain viable in the sample S.In this way, only the antibiotic resistant bacteria, for example, targetbacteria 180 are able to produce a detectable signal.

In some embodiments, the signal produced by the reporter molecules 130is independent of the quantity of the transduction particles 160 mixedwith the sample (when the quantity is above some de minimus value orpredetermined quantity). This is illustrated by FIGS. 7A and 7B. FIG. 7Ashows a schematic of the sample S containing the target bacteria 180,and that has been mixed with a series of transduction particles 160. Aportion of the transduction particles 160 are bound to the targetbacteria cells 180 in the sample S, such that the engineered nucleicacid 170 is conveyed into the target bacteria cells 180, as describedabove. The engineered nucleic acid 170 can then mediate the productionof reporter molecules (e.g., either via the viable cell reporter methodsor the gene reporter methods). After a first time period T1, a firstquantity of reporter molecules 130 is produced by the target bacteria180. As shown in FIG. 7B, after a second time period T2 greater than thefirst time period, the number of reporter molecules 130 increases to asecond quantity, greater than the first quantity. Because the reportermolecules 130 are not tagged by and/or contained within the transductionparticles 160, the amount of reporter molecules is substantiallyindependent of the amount of transduction particles 160 initially mixedwith the sample S. Moreover, as shown in FIG. 7B, the transductionparticles 160 are non-replicative, and there is no increase in thequantity of transduction particles 160 between the first time period T1and the second time period T2. Because the transduction particles 160are incapable of lytic replication, there is no decrease in the quantityof target bacteria.

FIG. 8 is a flow chart of a method 200 of identifying viable bacteria inbiological samples, according to an embodiment. The method 200 can beperformed using any of the containers described herein (e.g., container1700, 2700, 3700, 4700 or the like), and any of the instruments (e.g.,instrument 1100, 11000) and/or any components shown and describedherein. The method 200 can also be performed using any of thetransduction particles and/or viral vectors described herein, such as,for example, the transduction particle 160. More particularly, theoperations of the method 200 described below can be performed in acontainer, without exposing the samples, and/or reagents to outsideconditions. For purposes of the description, the method 200 is describedas being performed with the container 1700 and instrument 1100, shownand described above with reference to FIGS. 2-3.

The method 200 includes communicating a collected sample that cancontain target bacteria, for example, a patient nasal swab, to acontainer, 202. In some embodiments, this operation can be optional. Forexample, in some embodiments, the container can be received by a userwith the sample predisposed therein. The container can be any suitablecontainer, such as, for example, the container 1700 or any othercontainer described herein. The container can have a solution disposedin an interior region of the container. The solution can include, forexample, a bacterial nutrient media, buffers, surfactants or any othercomponent to facilitate growth of the target bacteria, production ofreporter molecules within the target bacteria, detection of bacteria orthe like. In some embodiments, the solution can be added after thesample is conveyed into the container. In some embodiments, the solutioncan be predisposed in the container, isolated from the sample, e.g., ina separate compartment in the container or the cap of the container suchthat it can be communicated to the sample on demand and/or in aclosed-system environment.

The container is then sealed, 204, for example, with a cap, reagentmodule or the like. In some embodiments, the seal can be formed by areagent module (see e.g., the reagent modules 3740 and 4740 describedbelow), that can include compartments, frangible portions, reagents,actuators, and/or nozzles. In some embodiments, an antibiotic or aseries of antibiotics is optionally added to the sample disposed in thecontainer, 206. The antibiotics can be selected and/or formulated tokill other non-targeted bacterial strains, for example, non-drugresistant strains, so that only the drug resistant strain survives. Inthis manner, the reporter molecules produced are necessarily produced bythe remaining, targeted bacterial strains. In some embodiments, theantibiotic/series of antibiotics can be predisposed in the container(for example, in the solution). In other embodiments, theantibiotic/series of antibiotics can be disposed in a separatecompartment (e.g., in the body or cap of the container assembly), andcan be communicated into the sample solution on demand or at apredetermined time.

A first reagent or substance containing the transduction particles, iscommunicated into and/or mixed with the sample, 208. The transductionparticles can be any of the transduction particles described herein,such as, for example the transduction particle 160. In particular, thetransduction particles include a nucleic acid molecule that contains areporter sequence that provides instructions for the target bacteria toexpress a reporter molecule. In some embodiments, the transductionparticles need not be added to the solution, but rather are predisposedin the solution. In such embodiments, the transduction particles aremixed with the sample to enable identification of the target bacteriumby the transduction particles. In some embodiments, for example, thetransduction particles are disposed in a separate compartment of the capof the container, and are released and/or mixed with the sample ondemand or at a predetermined time.

In some embodiments, the container solution can optionally be agitated,210 to efficiently mix the transduction particles and the solution tofacilitate interaction. Methods of mixing can include vortexing, manualshaking, stirring or the like, automated agitation (e.g., via a shakertable), or any other suitable agitation method. The container is thenplaced in the instrument or a portion of an instrument, 212, andmaintained for a predefined time and/or at a predetermined temperature,214. Such conditions can include, for example maintaining the sample forless than 2 hours, approximately 2 hours, 4 hours, 6 hours, 8 hours, upto 18 hours, or even more, at temperature, e.g., less than or equal toapproximately 37 degrees Celsius. The conditions under which the sampleis maintained are defined to allow the transduction particle to bind tothe target bacteria and communicate engineered nucleic acid to thetarget bacteria, and to promote the expression of the reporter molecules(e.g., luciferase). In the viable cell reporter embodiment, theengineered nucleic acid can be introduced to all of the target bacteriairrespective of, for example, the presence of a gene imparting drugresistance in the bacteria. Further specificity can be achieved, forexample, by eliminating all other phenotypes imparted by particulargenotypes, e.g., by adding anti-biotic to the sample as described abovewith reference to operation 206, and thereby eliminating or otherwisepreventing the generation of a signal from cells lacking a drugresistance gene and/or expressed drug resistant genotype.

In some embodiments, a reagent is then communicated into the sample toenhance, catalyze and/or promote the production of a signal from thereporter molecules, 216. For example, the reagent can be a substrateformulated to catalyze the production of a light signal by the reportermolecules. Such substrates can include an active ingredient, forexample, tridecanal that can interact with the reporter molecule toproduce a detectable signal, for example, luminescence. In someembodiments, the substrate can include a 6-carbon aldehyde (hexanal), a13-carbon aldehyde (tridecanal) and/or a 14-carbon aldehyde(tetradecanal), inclusive of all the varying carbon chain lengthaldehydes therebetween. In some embodiments, the reagent can beformulated to include Tween 20 or other surfactants, tridecanal or otheraldehydes, and adjusted to a particular pH. In some embodiments, thesubstrate can be stored in the container, for example, in an isolatedcompartment in the container cap, and can be delivered to the sample ondemand or at predetermined time.

The signal is detected, 218, using any suitable the detector. Thedetector can be, for example, the detector 1200 disposed in theinstrument 1100 or the detector (PMT) 11200 shown and described below.If a measureable signal is detected, then the target bacteria arepresent in the sample, 220. If no signal is detected, then the sample isfree of target bacteria, 222.

Although, the method 200 shown and described above can be used fordetection of viable bacteria and identification (e.g., by optionallyincluding antibiotics to eliminate non-targeted strains), in otherembodiments, methods can include transduction particles and/orengineered viral vectors that can selectively identify and/or recognizea bacteria by genotype. Said another way, the transduction particles canconditionally produce reporter molecules only after recognizing and/oridentifying a gene sequence within the target bacteria, as describedbelow.

The transduction particle can be engineered to encapsulate and deliveran engineered nucleic acid molecule, such as the nucleic acid molecule170, into the target bacteria. In some embodiments, the nucleic acidmolecule is engineered and/or formulated to include a nucleic acidsequence that is homologous to a target gene, a reporter gene, and anyother suitable regulatory genes. The recognition gene can be, forexample, homologous to a portion of the target bacteria gene andconfigured to probe for and recognize the portion of the bacterial gene,and operably couple itself to the bacterial gene and result in theintegration of the reporter gene within the target gene locus, e.g.,using homologous recombination. In some embodiments, the reporter genecan be a promoter-less gene and thus is only expressed if it becomesoperatively linked to a target gene promoter after insertion of thereporter gene into a target gene locus.

In some embodiments, an engineered nucleic acid molecule can beformulated and/or configured to conditionally recombine in the presenceof a target gene present in and/or specific to particular drug-resistantbacteria genotype. For example, FIG. 9 is a schematic illustration offormulation of an engineered nucleic acid molecule 570 and/or plasmidspecific for MRSA, according to an embodiment. The engineered nucleicacid sequence 500 includes plasmid regulatory elements 576 (e.g. anorigin of replication, etc.), a mecA gene fragment 572, a luxA gene 574a, a luxB gene 574 b, and a selectable marker (e.g. tetracyclineresistance gene, etc.) (not shown). The plasmid regulatory elements 576can be any plasmid regulatory element as known in the arts. The mecAgene fragment 572 is homologous to a segment of the mecA gene. Onceinside the bacteria, the mecA gene fragment 572 can probe for and/oridentify the mecA sequence on the MRSA gene, and mediate the integrationof the reporter genes into the mecA gene locus via homologousrecombination in a manner that operatively links the mecA gene promoterto the luxAB genes. In some embodiments, the engineered nucleic acid 500can include any other recognition sequence, e.g., tcdB, vanA, etc.,specific for any gene sequence on any other bacteria, for example, E.coli, Salmonella, C. difficile, VRE, etc. The luxA 574 a and luxB 574 bgenes, together serve as the reporter gene that can be controlled by thenatural bacteria transcription and translation cycle to express thereporter molecule luciferase. In some embodiments, any other reportergene can be used, for example, a gene for expressing an enzyme (e.g.,glucose oxidase, horseradish peroxidase) or a fluorescent protein (e.g.,green fluorescent protein, etc.).

FIG. 10 is a flow chart of a method 300 for genotypic identification ofbacteria using a transduction particle and/or engineered viral vector,e.g., transduction particle 160 that contains an engineered nucleic acidmolecule (e.g., the nucleic acid molecule 570). The method 300 can beperformed using any of the containers described herein, for example,container 1700, 2700, 3700, 4700 and the like, and any instruments andcomponents thereof described as herein, such as, for example instruments1100 and 11000.

The method 300 includes adding a transduction particle to a sample, e.g.a patient nasal swab that can contain target bacteria genotype, 302. Thesample can be disposed in a container, such as, for example, container1700, and can further include solutions such as, for example, bacterialnutrient media, buffers, and/or surfactants. In some embodiments, thetransduction particle can be included in the solution and disposed inthe container prior to adding the sample. The transduction particles andthe solution are maintained under conditions such that the transductionparticles identify and bind to the target bacteria present in thesample, 304. The transduction particle then inserts the engineeredplasmid or engineered nucleic acid molecule into the target bacteria,306.

The recognition gene portion of the engineered nucleic acid molecule,for example, a mecA gene fragment, as described herein, then probes thebacteria DNA for a homologous sequence, 308. If the homologous sequenceis present, the engineered nucleic acid molecule inserts into andoperatively couples the reporter gene sequences with an endogenouspromoter of the target gene 310. In this manner, the target bacteriaexpress reporter molecules, for example luciferase, through its naturaltranscription/translation process, 312.

The sample is then maintained for a predefined time and/or at apredetermined temperature, 314. Such conditions can include, for examplemaintaining the sample for less than 2 hours, approximately 2 hours, 4hours, 6 hours, 8 hours, up to 18 hours, or even more, at temperature,e.g., less than or equal to approximately 37 degrees Celsius. Theconditions under which the sample is maintained are defined to allow thetransduction particle to bind to the target bacteria and communicateengineered nucleic acid to the target bacteria, and to promote theexpression of the reporter molecules (e.g., luciferase).

In some embodiments, a reagent is then communicated into the sample toenhance, catalyze and/or promote the production of a signal from thereporter molecules, 316. For example, the reagent can be a substrateformulated to trigger the production of a light signal by the reportermolecules. Such substrates can be any suitable substrates of the typesshown and described herein. The signal is detected, 318, with anysuitable instrument. If the sample contained any other bacteriagenotype, the gene sequence homologous to the recognition gene would notbe present in the bacteria DNA. Accordingly, there would be nohomologous recombination of transduction particle DNA with bacteria DNAand the reporter molecule would not be produced. Therefore, addingsubstrate to the sample solution 316 would not produce any detectablesignal indicating that the sample does not contain the target bacteriagenotype.

FIGS. 11A, 11B, 11C, and 11D show a schematic illustration of portionsof the method 300 for genotypic identification and detection of a targetbacteria. As shown in FIG. 11A, a transduction particle 660 added toand/or mixed with a sample S to detect a particular genotype of a targetbacteria 680 that may be present in the sample S. In the first operation(indicated by the schematic illustration 692), the transduction particle660 is brought into contact with the sample S containing the targetbacteria 680. The transduction particle 660 can specifically identifyand bind to the target bacteria 680 cell wall as shown by arrow C. Thetransduction particle 660 then communicates an engineered nucleic acidor plasmid 670 contained therein, into the target bacteria 680cytoplasm, as shown by arrow D (see operation 694).

The engineered nucleic acid 670 can be any of the engineered nucleicacid molecules described herein, such as, for example, the nucleic acidmolecule 570. In particular, the engineered nucleic acid molecule 670includes a recognition sequence 672, engineered and/or formulated torecognize a target gene 684 in the DNA 682 of the target bacteria 680.The engineered nucleic acid also includes a reporter gene sequence 674(e.g., the reporter sequence luxA/luxB). If the target bacteria 680contains the target gene 684, the engineered nucleic acid 670 willrecognize the target gene 684 and inserts the reporter gene sequence 674into the target gene loci in a manner that operatively links thereporter gene sequences 674 with a target gene 682 promoter (seeoperation 696). For example, the engineered nucleic acid 670 can includea recognition sequence 672 specific for the mecA sequence on MRSA DNA.In the final operation (see schematic 698), the transcription andtranslation machinery of the target bacteria 680 read the genes encodingthe reporter sequence 674 and produce the reporter molecules 630. If thetarget gene 684 is not present, recombination does not take place andthe reporter molecules 630 are not expressed. Therefore, presence of thereporter molecules 630 indicates that the target gene is present in theviable bacteria 680 in the sample S. Since the transduction particle 660is non-replicative and is incapable of lytic or lysogenic replication,the target bacteria 680 remain alive during the assay. Therefore, thesystems and methods described herein can be used to detect genes withinlive bacteria.

In some embodiments, a system 1000 and/or any of the methods disclosedherein can include and/or be performed with a container configured tofacilitate communication of a solution (e.g., nutrient media, buffers,surfactants), transduction particles, biologic vectors such asengineered viral vector, abiologic vectors consisting of polymers,liposomes or virus-like particles, and/or reagent (e.g., substrate,antibiotics, etc.) into the sample. For example, FIGS. 12-14 show acontainer assembly 1700 according to an embodiment, in a firstconfiguration, a second configuration and a third configuration,respectively. One or more container assemblies 1700 can be disposedwithin any suitable instrument of the type disclosed herein (see e.g.,instrument 11000 described below) configured to manipulate, actuateand/or interact with the container assembly 1700 to perform the methodsassociated with the identification of the target cell described herein.The container assembly 1700 allows for efficient and accurate diagnostictesting of samples by limiting the amount of sample handling duringassay. Moreover, modular arrangement of the container components (e.g.,the reaction chamber 1732 and the reagent module 1740) allows any numberof different reagent modules 1740, each containing different reagentsand/or formulations to be interchangeably used to detect a differenttype of target cell. This arrangement also allows the transductionparticles and the reagents to be stored separately and communicated tothe sample on demand, as described below. Separate storage can beuseful, for example, if the reagents included within the reagent module1740 have different storage requirements (e.g., expiration dates,lypophilization requirements, storage temperature limits, etc.) than thereagents, solution and/or sample included within the reaction chamber1732.

As shown, the container assembly includes a reaction chamber 1732 and areagent module 1740. The reaction chamber 1732 can be coupled to thereagent module 1740 to form an integrated assembly. The reaction chamber1732 can be formed from any suitable material, such as, for example,light weight, rigid and inert materials (e.g., plastics). At least aportion of the reaction chamber 1732 can be at least partiallytransparent to allow viewing and/or detection of the internal volume ofthe reaction chamber 1732 (for example, to view luminescence in thereaction chamber 1732). In some embodiments, the reaction chamber 1732can be shaped as a cylinder with a rounded bottom, or flat base. Inother embodiments, the reaction chamber 1732 can have any other suitableshape, e.g., square, rectangular, oval, polygonal, etc. In someembodiments, the reaction chamber 1732 can have a diameter of 12 mm anda height of 75 mm. In some embodiments, the diameter of the reactionchamber 1732 can be sized to optimally match the cross-section of adetector (e.g., detector 1200, or 11212 included in the instrument11000, or any other detector). In some embodiments, the containerassembly 1700 can be provided with one or more solutions and/or reagentsof the types shown and described herein (e.g., bacterial nutrientsolution, buffers, surfactants, transduction particles, and/orantibiotics), predisposed within the reaction chamber 1732.

The reagent module 1740 of the container 1700 includes a housing 1741, afirst actuator 1750 and a second actuator 1760. The housing 1741 isconfigured to be removably coupled to the reaction chamber 1732 by anysuitable mechanism. For example, in some embodiments, the housing 1741can be coupled to the reaction chamber 1732 by a threaded coupling, aninterference fit, snap-fit, or the like. In some embodiments, thehousing 1741 and the reaction chamber 1732 define a substantially fluidtight seal.

The housing 1741 defines a first reagent volume 1742 and a secondreagent volume 1744. The first reagent volume 1742 and the secondreagent volume 1744 can be separated by a sidewall 1746. In someembodiments, the first reagent volume 1742 contains biologic orabiologic vectors, transduction particles and/or a viral vector (e.g.,transduction particle 110, 160 or any of the other transductionparticles described herein), that includes an engineered nucleic acidmolecule (e.g., engineered nucleic acid 170) formulated to cause thetarget cell (e.g., bacteria) to produce a series of reporter molecules(e.g., luciferase). In some embodiments the transduction particle isformulated to be non-replicative (i.e., incapable of replication), asdescribed herein. In some embodiments, the second reagent volume 1744can contain a reagent formulated to interact with the reporter moleculeto catalyze, enhance the production of and/or produce a measurablesignal, such as, for example an optical signal. In some embodiments, thereagent is a luciferase substrate of the types shown and describedherein, such as, for example, a composition including tridecanal.Although the transduction particles and the reagent are shown as beingdisposed directly within the first reagent volume 1742 and the secondreagent volume 1744, respectively, in other embodiments, thetransduction particles and/or reagents can be disposed inside reagentcontainers (not shown) shaped and sized to be disposed substantiallyinside the first reagent volume 1742 and/or the second reagent volume1744.

In some embodiments, the housing 1741 and/or any reagent containerstherein (not shown) can include frangible portions configured to rupturewhen actuated or compressed (e.g., by the first actuator 1750 and/or thesecond actuator 1760). In this manner, the reagents and constituents canbe stored in isolation and released upon actuation. In some embodiments,the housing 1741, first actuator 1750, second actuator 1760 and/or suchreagent containers can include features to facilitate repeatabledelivery, e.g., curved edges, flat bottom, bellowed walls, or any othersuitable feature. In some embodiments, for example, the housing 1741 caninclude a puncturer or a series of puncturers (not shown) disposedwithin the first reagent volume 1742, configured to puncture a portionof a reagent container when the plunger portion 1754 of the firstactuator 1750 is moved within the first reagent volume 1742. In someembodiments, a puncturer can also be disposed within the second reagentvolume 1744.

The first reagent volume 1742 and the second reagent volume 1744 canhave any suitable shape, orientation and/or size. In some embodiments,as shown in FIGS. 12-14, the first reagent volume 1742 and the secondreagent volume 1744 can be concentric. In other embodiments, the firstreagent volume 1742 and the second reagent volume 1744 can be locatedparallel to each other. Although shown as having a substantiallyconstant cross-sectional area, in some embodiments, a cross-sectionaldimension, e.g., diameter or area, of the housing 1741 and/or the firstreagent volume 1742 and the second reagent volume 1744 can be varied toincrease/decrease a volume of the reagent contained therein. In thismanner, the housing 1741 can be configured to provide the desiredquantity and/or a flow rate of reagent to be delivered into the reactionchamber 1732.

The housing 1741 includes a delivery portion 1770 that, when the housing1741 is coupled to the reaction chamber 1732, defines a first fluidicpathway 1772 a between the first reagent volume 1742 and the reactionchamber 1732, and a second fluidic pathway 1772 b between the secondreagent volume 1744 and the reaction chamber 1732. As shown, the firstfluidic pathway 1772 a and the second fluidic pathway 1772 b areseparate from each other. The first fluidic pathway 1772 a and thesecond fluidic pathway 1772 b include a first outlet 1774 a and a secondoutlet 1774 b, respectively, that each open into the reaction chamber1732. The first fluidic pathway 1772 a and the second fluidic pathway1772 b provide a pathway for the transduction particles and reagentsdisposed in the first reagent volume 1742 and/or the second reagentvolume 1744, respectively, to be communicated into the reaction chamber1732.

The delivery portion 1770 can be configured to provide any suitablepathway and/or mechanism for delivering the transduction particles andreagents disposed in the first reagent volume 1742 and/or the secondreagent volume 1744 into the reaction chamber 1732. For example, in someembodiments, the delivery portion 1770 can include a single fluidicpathway for communicating fluids from the first reagent volume 1742 andthe second reagent volume 1744 into the reaction chamber 1732. In someembodiments, the delivery portion 1770 can be configured to deliverreagents from the first reagent volume 1742 and/or the second reagentvolume 1744 into the reaction chamber 1732 in a manner that promotesmixing and/or that minimizes aeration, overspray and/or undesirableturbulence. In some embodiments, the first fluidic pathway 1772 a and/orthe second fluidic pathway 1772 b can have a varying cross-sectional (orflow) areas (e.g., the pathways can resemble nozzles) to produce acontrolled flow rate of the substances flowing therethrough. In someembodiments, the flow rate of the transduction particles and/orreagents, can be 1 ml/sec, 2 ml/sec,3 ml/sec, 4 ml/sec, 5 ml/sec, or anysuitable flow rate to sufficiently mix the substance and/or to minimizeaeration.

The reagent module 1740 includes the first actuator 1750 at leastpartially disposed in the housing 1741. The first actuator 1750 includesan engagement portion 1752 and a plunger portion 1754, which is disposedwithin the first reagent volume 1742. The engagement portion 1752 of thefirst actuator 1750 is configured to be manipulated (for example, by anyinstrument shown and described herein, such as the instrument 11000) tomove the plunger portion 1754 within the first reagent volume 1742. Insome embodiments, the plunger portion 1754 of the first actuator 1750and a portion of the housing 1741 define and/or include a seal tofluidically isolate the first reagent volume 1742 from a volume outsideof the housing 1741. In some embodiments, the seal can be, for example,a gasket, an o-ring, a rubber seal, or any suitable seal. As shown, theengagement portion 1752 and the plunger portion 1754 of the firstactuator 1750 can have different cross-sectional dimensions (e.g.,diameter). In other embodiments, however, the engagement portion 1752and the plunger portion 1754 of the first actuator 1750 can have thesame cross-sectional dimension (e.g., diameter). In some embodiments,the engagement portion 1752 can be offset from (e.g., non-coaxial with)the plunger portion 1754.

The reagent module 1740 includes the second actuator 1760 at leastpartially disposed in the housing 1741. The second actuator 1760includes an engagement portion 1762 and a plunger portion 1764, which ismovably disposed within the second reagent volume 1744. The engagementportion 1762 of the second actuator 1760 is configured to be manipulated(for example, by any instrument shown and described herein, such as theinstrument 11000) to move the plunger portion 1764 of the secondactuator 1760 within the second reagent volume 1744. In someembodiments, the plunger portion 1764 of the second actuator 1760 and aportion of the housing 1741 define and/or include a seal to fluidicallyisolate the second reagent volume 1742 from a volume outside of thehousing 1741. In some embodiments, the seal can be, for example, agasket, an o-ring, a rubber seal, or any suitable seal, and can besubstantially similar to the seal of the first actuator 1750. As shown,the engagement portion 1762 and the plunger portion 1764 of the secondactuator 1760 can have different cross-sectional dimensions (e.g.,diameter). In other embodiments, however, the engagement portion 1762and the plunger portion 1764 of the second actuator 1760 can have thesame cross-sectional dimension (e.g., diameter). In some embodiments,the engagement portion 1762 can be offset from (e.g., non-coaxial with)the plunger portion 1764.

As shown, the engagement portion 1762 of the second actuator 1760 atleast partially surrounds the engagement portion 1752 of the firstactuator 1750. Similarly stated, at least a portion of the firstactuator 1750 and a portion of the second actuator 1760 are disposedconcentrically in the housing 1741. In this manner, the reagent module1740 can be coupled to the reaction chamber 1732 and/or disposed in aninstrument in any angular orientation about the longitudinal axis of thecontainer assembly 1700. This arrangement allows a single actuatorassembly to manipulate both the first actuator 1750 and the secondactuator 1760.

More particularly, the second actuator 1760 defines a channel 1766within which the engagement portion 1752 of the first actuator 1750 canmove when the first actuator 1750 is manipulated to move the plungerportion 1754. In some embodiments the engagement portion 1762 of thesecond actuator 1760 can define an opening within which the engagementportion 1752 of the first actuator 1750 can be substantially disposed.Although a longitudinal axis of the plunger portion 1754 of the firstactuator 1750 is shown as being concentric to a longitudinal axis of theplunger portion 1764 of the second actuator 1760, in other embodiments,the longitudinal axis of the plunger portion 1754 can be offset fromand/or nonconcentric with the longitudinal axis of the plunger portion1764. In some embodiments, for example, the first actuator 1750 and thesecond actuator 1760 can be disposed adjacent but not concentric to eachother. In some embodiments, the first actuator 1750 and the secondactuator 1760 can be disposed parallel to each other. In someembodiments the engagement portions 1752 and/or the engagement portion1762 can be recessed in the housing 1741, e.g., to prevent accidentalactuation.

The first actuator 1750 and the second actuator 1760 can be moved in anysuitable manner to perform the functions described herein. For example,in some embodiments, the plunger portion 1754 of the first actuator 1750can be moved independently of the movement of the plunger portion 1764of the second actuator 1760. In some embodiments, the first actuator1750 and second actuator 1760 can have the same stroke length. In otherembodiments, the first actuator 1750 and second actuator 1760 can havedifferent stroke lengths. In this manner, different volumes (and/ordifferent flow rates) of transduction particles or reagents can beconveyed into the reaction chamber 1732.

In use, the container assembly 1700 is configured to be manipulated toperform the methods and/or assays described herein while maintainingfluidic isolation of the reaction chamber 1732. Similarly stated, thereaction chamber 1732 and the reagent module 1740 of the containerassembly 1700 can collectively define a closed system within whichtarget cell identification can be performed (i.e., without decouplingthe reagent module 1740 from the reaction chamber 1732). In particular,the container assembly 1700 can be delivered to the user in a firstconfiguration (FIG. 12), in which the first actuator 1750 and the secondactuator 1760 are each their respective first positions. Although thereagent module 1740 is shown as being coupled to the reaction chamber1732 in FIG. 12, the reagent module 1740 can initially be decoupled fromthe reaction chamber 1732 to allow a sample containing the target cell(e.g., bacteria) to be disposed in the internal volume defined by thereaction chamber 1732. The reagent module 1740 can be coupled to thereaction chamber 1732 to define a fluid-tight seal.

To move the container assembly 1700 and/or the reagent module 1740 tothe second configuration (FIG. 13), the engagement portion 1752 of thefirst actuator is manipulated to move within the channel 1766. In thismanner, the plunger portion 1754 of the first actuator 1750 moves withinthe first reagent volume 1742 to convey and/or expel the reagent (e.g.,transduction particles) from the first reagent volume 1742 through thefirst fluidic pathway 1742 a and first outlet 1744 a, and into thereaction chamber 1732 as shown by arrow EE. In some embodiments, thereagent includes transduction particles that interact with the targetcell contained in the sample such that the target cells produce a seriesof reporter molecules according to any of the methods described herein.Manipulation and/or maintaining of the container assembly 1700 (and thesample contained therein) can be performed by the instruments 1100and/or 11000 as described herein and/or any other instruments orcomponents described herein.

To move the container assembly 1700 and/or the reagent module 1740 tothe third configuration (FIG. 14), the engagement portion of 1762 of thesecond actuator 1760 is manipulated to move at least partially about thefirst actuator 1750. The movement of the engagement portion 1762 movesthe plunger portion 1764 of the second actuator 1760 from the firstposition to a second position within the second reagent volume 1744. Thedisplacement of the plunger portion 1764 conveys and/or expels thereagent (e.g., a substrate such as tridecanal) from within the secondreagent volume 1744 through the second fluidic pathway 1772 b and secondoutlet 1774 b into the reaction chamber 1732, as shown by arrow FF. Insome embodiments, the reagent is a substrate that can interact with thereporter molecules produced to urge, catalyze and/or enhance thereporter molecules to produce a signal, e.g., via a luminescencereaction.

In some embodiments, a container assembly can include a mechanism forcollecting a sample and/or disposing a sample into the containerassembly. For example, in some embodiments, a sample can be collectedusing a swab, which is then disposed into the reaction chamber of thecontainer. FIGS. 15-17 show a container assembly 2700 in a firstconfiguration, a second configuration, and a third configuration,respectively. The container assembly 2700 includes a reaction chamber2732 and a temporary cap 2739. The reaction chamber 2732 of thecontainer assembly 2700 can be coupleable to any reagent module, suchas, for example, the reagent module 1740 as described above, or anyother reagent module described herein.

The sample S containing the target cell can be collected in a swab 2734,which can be a nasal swab, saliva swab, environmental swab, or the like.In some embodiments, after collecting the sample S, the nasal swab 2734is broken at a predefined position and/or length of the swab 2734 asshown by the line GG (FIG.15). The reaction chamber 2732 can contain asolution 2702, e.g., nutrient media, buffers, surfactants, and/or anyother reagents formulated to maintain the target cells, promote theproduction of reporter molecules or the like. The swab 2734 is insertedinto the reaction chamber 2732, as shown by arrow HH, until it isimmersed in the solution 2702.

The temporary cap 2736 can then be coupled to the reaction chamber 2732to place the container assembly 2700 in the second configuration (FIG.16). In some embodiments, the temporary cap 2736 can define asubstantially fluid-tight seal when coupled to the reaction chamber2732. In this manner, the container assembly 2700 can be agitated, e.g.,vortexed or shaken, to allow a significant portion of the sample Scontaining the target cells to communicate into the solution 2702 fromthe swab 2734. In some embodiments, the swab 2734 and the samplecollection protocol can be defined such that as much as 50%, 60%, and upto 70%, and any quantity therebetween or even higher, of the collectedtarget cells are transferred into the solution 2702.

In some embodiments, the swab 2734 can be removed for testing. Removalof the swab, in certain situations, can limit interference with samplemeasurements. Accordingly, in some embodiments, the temporary cap 2736can include a gripping mechanism, e.g., notches, grooves, sleeve, or anyother feature for gripping the swab 2734. In such embodiments, when thetemporary cap 2736 is removed from the reaction chamber 2732 as shown inthe third configuration by arrow II (FIG. 17), the swab 2734 is alsoremoved with it. In some embodiments, the reaction chamber 2732 caninclude one or more labels 2738 disposed on an exterior surface of thereaction chamber 2732. The labels 2738 can include informationassociated with the container assembly 2700, e.g., target cell, serialnumber, lot number, expiration date, and/or warning information. In someembodiments, the container 2700 can also include a tracking mechanism2739, e.g., bar codes on labels and/or RFID tags.

FIGS. 18-25 show a container assembly 3700 according to an embodimentthat includes a reaction chamber 3732 and a reagent module 3740 that iscoupleable to the reaction chamber. The container assembly 3700 can beused with and manipulated by any of the instruments described herein,e.g., instrument 11000, and/or any of the components described herein.The container assembly 3700 can also be used to perform any of themethods described herein, e.g., such as the methods 150, 200 and 300described above.

The reaction chamber 3732 is configured to contain a sample and/or otherreagents, and can be formed from a light weight, rigid and inertmaterial. At least a portion of the reaction chamber 3732 (e.g., thedistal end portion) can be at least partially transparent to allowviewing, optical access and/or detection of the internal volume of thereaction chamber 3732. Although shown as being shaped as a cylinder witha rounded bottom, in other embodiments, the reaction chamber 3732 canhave any other suitable shape, e.g., square, rectangular, oval,polygonal, etc. In some embodiments, the reaction chamber 3732 can havea diameter of 12 mm and a height of 75 mm. In some embodiments, thecontainer assembly 3700 can be provided with one or moresolutions/reagents (e.g., bacterial nutrient solution, buffers,surfactants, transduction particle, and/or antibiotics), predisposedwithin the reaction chamber 3732.

The reagent module 3740 includes a housing 3741, a first actuator 3750,a second actuator 3760, a delivery portion 3770, a first reagentcontainer 3780 a and a second reagent container 3780 b. As shown in FIG.18, the housing 3741 is configured to be removably coupled to thereaction chamber 3732 by any suitable mechanism. For example, in someembodiments, the housing 3741 can be coupled to the reaction chamber3732 by a threaded coupling, an interference fit or the like. In someembodiments, the housing 3741 and the reaction chamber 3732 define asubstantially fluid tight seal.

FIG. 20 shows a top view of the housing 3741. The housing 3740 defines afirst reagent volume 3742 and a second reagent volume 3744 that can beseparated by a sidewall 3746. The housing 3741 can be formed from alightweight and rigid material, such as, for example, injection moldedplastic. In some embodiments, the housing 3741 can have a diameter ofapproximately 24 mm. In some embodiment, the diameter of the housing3741 can be varied to increase or decrease the capacity of the firstreagent volume 3742 and the second reagent volume 3744. In someembodiments, the diameter of the first reagent volume 3742 and/or thesecond reagent volume 3744 can be varied (i.e., not equal to eachother).

As shown in the top view of the housing 3741 in FIG. 20 and inclinedbottom view of the housing 3741 shown in FIG. 21, the housing 3741includes a delivery portion 3770 that, when the housing 3741 is coupledto the reaction chamber 3732, defines a first fluidic pathway 3772 abetween the first reagent volume 3742 and the reaction chamber 3732, anda second fluidic pathway 3772 b between the second reagent volume 3744,and the reaction chamber 3732. The first fluidic pathway 3772 a and thesecond fluidic pathway 3772 b include a first outlet 3774 a and a secondoutlet 3774 b, respectively, that open into the reaction chamber 3732,when the reaction chamber 3732 is coupled to the housing 3741. The firstfluidic pathway 3772 a (and the outlet 3774 a) and the second fluidicpathway 3772 b (and the outlet 3774 b) provide a pathway for reagentsdisposed in the first reagent volume 3742 and the second reagent volume3744 to be communicated into the reaction chamber 3732. Although thefirst fluidic pathway 3772 a and the second fluidic pathway 3772 b areshown as being separate from each other, in other embodiments, the firstfluidic pathway 3772 a and the second fluidic pathway 3772 b can includea common boundary and/or be in fluid communication with each other.

The delivery portion 3770 is configured to provide any suitable pathwayand/or mechanism for delivering the transduction particles and reagentsdisposed in the first reagent volume 3742 and/or the second reagentvolume 3744 into the reaction chamber 3732. For example, in someembodiments, the first fluidic pathway 3772 a and the second fluidicpathway 3772 b can be configured to deliver reagents from the firstreaction volume 3742 and the second reaction volume 3744, respectively,to the reaction chamber 3732 in a manner that promotes mixing and/orminimizes aeration, overspray and/or undesirable turbulence. The firstfluidic pathway 3772 a and the second fluidic pathway 3772 b canaccommodate any suitable flow rate, e.g., 1 ml/sec, 2 ml/sec, 3 ml/sec,4 ml/sec, 5 ml/sec. In some embodiments, at least a portion of thedelivery portion 3770, can be disposed within the reaction chamber 3732when the housing 3741 is coupled to the reaction chamber 3732.

As shown in FIG. 19 and the side cross-section of the container shown inFIG. 23 the reagent module 3740 includes the first actuator 3750disposed in the housing 3741. The first actuator 3740 includes anengagement portion 3752 and a plunger portion 3754, which is movablydisposed within the first reagent volume 3742. When actuated, theengagement portion 3752 of the first actuator 3750 moves the plungerportion 3754 within the first reagent volume 3742. As shown, the plungerportion 3754 of the first actuator 3750 includes a seal 3769 a tofluidically isolate the first reagent volume 3742 from a volume outsideof the housing 3741. In some embodiments, the seal 3769 a can be, forexample, a gasket, an o-ring, a rubber seal, or any suitable seal.

The reagent module 3740 includes the second actuator 3760. The secondactuator 3760 includes an engagement portion 3762 and a plunger portion3764 that is movably disposed within the second reagent volume 3744.When actuated, the engagement portion 3762 of the second actuator 3760moves the plunger portion 3764 of the second actuator 3760 within thesecond reagent volume 3744. The plunger portion 3764 of the secondactuator 3760 includes a seal 3769 b to fluidically isolate and/orprevent leakage of any reagent contained in the second reagent volume3744.

The first actuator 3750 and the second actuator 3760 can be disposed ina nested configuration in the housing 3741. Said another way, the firstactuator 3750 and the second actuator 3760 can be disposedconcentrically, such that the first actuator 3750 is nested within thesecond actuator 3760. In this manner, the reagent module 3740 can becoupled to the reaction chamber 3732 and/or disposed in an instrument inany angular orientation about the longitudinal axis of the containerassembly 3700. More particularly, the second actuator 3760 defines achannel 3766 within which the engagement portion 3752 of the firstactuator 3750 can move when the first actuator 3750 is manipulated tomove the plunger portion 3754. Further, the engagement portion 3762 ofthe second actuator 3760 defines an opening 3767 within which theengagement portion 3752 of the first actuator 3750 is substantiallydisposed (when the reagent module 3740 is in the first configuration orthe third configuration). A longitudinal axis of the plunger portion3754 of the first actuator 3750 is offset from (i.e., non-coaxial with)a longitudinal axis of the plunger portion 3764 of the second actuator3760. The plunger portion 3754 of the first actuator 3750 can be movedindependently of the movement of the plunger portion 3764 of the secondactuator 3760. Furthermore, the first actuator 3750 and the secondactuator 3760 can be recessed inside the housing, for example, toprevent accidently actuation of the first actuator 3750 and the secondactuator 3760.

The first reagent volume 3742 can include the first reagent container3780 a and the second reagent volume 3744 can contain the second reagentcontainer 3780 b. As shown in FIG. 22 (which only shows the firstreagent container 3780 a for clarity), the first reagent container 3780a (and the second reagent container 3780 b) includes a sidewall 3782 aand a frangible member 3784 a, that together define an internal volume3786 a. In some embodiments, the sidewall 3782 a can also be frangible.The internal volume 3786 a can be completely or partially filled with areagent. For example, in some embodiments, the first reagent container3780 a can contain transduction particles (e.g., transduction particles110, 160 or any other transduction particles described herein) thatincludes an engineered nucleic acid (e.g., engineered nucleic acid 170)formulated to cause the target cell (e.g., bacteria) to produce aplurality of reporter molecules. The second reagent container 3780 b cancontain a second reagent formulated react with the reporter molecules toenhance the production of a signal. For example, in some embodiments,the reagent is a substrate, such as tridecanal, that can interact withthe reporter molecule (e.g., luciferase), to produce a measurablesignal, e.g., via a luminescence reaction.

The reagent containers can be shaped and sized to be disposedsubstantially inside the first reagent volume 3742 and the secondreagent volume 3744. The housing 3741 include a first puncturer 3792 aand a second puncturer 3792 b disposed within the first reagent volume3742 and the second reagent volume 3744, respectively. The puncturersare configured to rupture the respective frangible portions of the firstreagent containers 3780 a and the second reagent container 3780 b whenthe plunger portion 3754 and the plunger portion 3764 are displacedwithin the first reagent volume 3742 and the second reagent volume 3744,respectively. In some embodiments, the reagent containers can includecurved edges (see e.g., the curved edge 3789 a) and a bottom portion(see e.g., the bottom portion 3788 a) that can be substantially flat.The flat bottom portion and the curved edges can allow for spreading ofthe compressive force applied by the first actuator 3750 and the secondactuator 3760 on the first reagent container 3780 a and the secondreagent container 3780 b, respectively, to ensure repeatable delivery.

The reagent containers can be constructed from materials that aresubstantially impermeable to and/or substantially chemically inert fromthe substance contained therein, e.g., transduction particle, substrate,antibiotics, buffers, surfactants, or any other reagent that can berequired for the detection assay. In this manner, the reagents can bestored in the reagent containers for extended periods of time. Forexample, the side wall 3782 a of the reagent container 3780 a can beformed from a flexible and inert material, e.g., blister plastic,aluminum foil, aluminum laminate or any other suitable material.Moreover, in some embodiments, the frangible member 3784 a can beconstructed from a material having certain temperature characteristicssuch that the desired properties and integrity of the frangible member3784 a are maintained over a certain temperature. For example, in someembodiments, it can be desirable to store the reagent container 3780 acontaining reagent or substrate in a refrigerated condition. In someembodiments, the frangible member 3784 a can be constructed from apolymer film, such as any form of polypropylene. In some embodiments,the frangible member 3784 a can be constructed from bi-axially orientedpolypropylene (BOP). In some embodiments, the frangible member 3784 acan be constructed from aluminum.

The reaction chamber 3732 and the reagent module 3740 of the containerassembly 3700 can collectively define a closed system within whichtarget cell identification can be performed (i.e., without decouplingthe reagent module 3740 from the reaction chamber 3732). The containerassembly 3700 and/or the reagent module 3740 can be moved betweenmultiple different configurations to transfer reagents and/or substancesfrom the first reagent chamber 3742 and the second reagent chamber 3744.In particular, FIGS. 23-25 show the reagent module 3740 in a firstconfiguration, a second configuration and a third configuration,respectively. The reaction chamber 3732 is not shown for clarity.

The container assembly 3700 can be delivered to the user in the firstconfiguration (FIG. 23), wherein the first actuator 3750 is in a firstposition and the second actuator 3760 is in a first position. The firstreagent volume 3742 includes the reagent container 3780 a containing thereagent (e.g., transduction particles), and the second reagent volume3744 contains the second reagent container 3780 b that includes areagent (e.g., tridecanal, formulated to react with the reportermolecules).

To move the container assembly 3700 to the second configuration (FIG.24), the engagement portion 3752 of the first actuator 3750 ismanipulated to displace the plunger portion 3754 of the first actuator3750 within the first reagent volume 3742 from the first position to asecond position. Similarly stated, the engagement portion 3752 movesdistally within the channel 3766 and/or the opening 3767 of the secondactuator 3760. In this manner, the plunger portion 3754 of the firstactuator 3750 applies a force on the bottom portion 3788 a of the firstreagent container 3780 a. This pushes the frangible portion 3784 a ofthe first reagent container 3780 a against the puncturer 3769 a, untilthe frangible portion 3784 a ruptures, releasing the reagent containedtherein e.g., transduction particle, into the first reagent volume 3742.Further displacement of the plunger portion 3754 of the first actuator3750 towards the second position decreases the internal volume 3786 a ofthe reagent container 3780 a and the first reagent volume 3742. Thiscommunicates the reagent e.g., transduction particle from the firstreagent volume 3742 through the first fluidic pathway 3772 a and thefirst outlet 3774 a of the delivery portion 3770, and into the reactionchamber 3732. As shown, the first actuator 3750 include a recess 3758configured to receive a portion of the puncturer 3792 a to prevent thepuncturer from damaging the first actuator 3750 and/or from limiting thetravel of the first actuator 3750 towards the second position. In someembodiments, the reagent, e.g., transduction particle can interact withthe target cell contained in the sample and urge the target cell toproduce the reporter molecule as described herein.

To move the container assembly 3700 to the third configuration (FIG.25), the engagement portion of 3762 of the second actuator 3760 ismanipulated to displace the plunger portion 3764 of the second actuator3760 from the first position to a second position within the secondreagent volume 3744. Similar to the second configuration, thedisplacement of the second actuator 3760 causes a puncturer 3792 b torupture the frangible portion 3784 b of the second reagent container3780 b and communicate the substrate contained therein (e.g.,tridecanal) through the second fluidic pathway 3772 b and second outlet3774 b into the reaction chamber 3732. The substrate can interact withthe reporter molecules and urge, enhance and/or catalyze the reportermolecule to produce a signal, e.g., via a luminescence reaction. Asshown, the second actuator 3760 include a recess 3768 configured toreceive a portion of the puncturer 3792 b to prevent the puncturer fromdamaging the second actuator 3760 and/or from limiting the travel of thesecond actuator 3760 towards the second position. In some embodiments,the reagent, e.g., transduction particles, can interact with the targetcell contained in the sample and urge the target cell to produce thereporter molecule as described herein.

Although the exit portions of the first fluidic pathway 3772 a and thesecond fluidic pathway 3772 b are shown as being substantially linear,and having a substantially constant flow area, in other embodiments, adelivery portion can define any suitable flow pathways through which thereagents, substances, transduction particles and the like can bedelivered. For example, in some embodiments, a delivery portion can beconfigured to deliver one or more reagents into a reaction chamber in amanner that promotes mixing, that minimizes aeration, overspray and/orundesirable turbulence.

For example, in some embodiments, a reagent module can be configured todeliver a substance containing transduction particles of the types shownand described herein into a reaction chamber in a manner thatefficiently mixes the transduction particles with the sample. Forexample, in those embodiments in which a swab (such as the swab 2734) isretained within the reaction chamber, a reagent module can include adelivery nozzle or other mechanism for delivering transduction particlesthat enhances removal of portions of the sample from the swab. In thismanner, the mechanism of delivery can enhance the performance of theassay by improving the mixture of the sample and the transductionparticles. Such mechanisms can include, for example, high pressure jetnozzles, angled nozzles, multiple flow paths from a single reagentchamber into a reaction chamber, or the like.

In other embodiments, a reagent module can be configured to deliver areagent formulated to enhance, catalyze or trigger the production of alight signal (e.g., a substrate of the types shown and described herein)into a reaction chamber in a manner that enhances the measurement of thelight signal. For example, in some embodiments, a method of detectingthe reporter molecules includes detecting the intensity (or strength) ofa luminescence reaction triggered by the addition of a substrate intothe sample in which reporter molecules have been expressed. Moreparticularly, in some embodiments, the expressed reporter molecules andthe substrate are collectively formulated to produce a flash reaction inresponse to the addition of the substrate to the sample. Flash reactionsare luminescence reactions in which a distinct peak intensity occursvery quickly after the addition of the substrate (e.g., substantiallyinstantaneously, within several seconds and/or less than one minute).Although flash reactions can produce very sensitive results (which arebeneficial for detection of small quantities, etc.), the accuratemeasurement of such transient reactions can be challenging. In contrast,glow reactions are longer lasting luminescence reactions characterizedby a stable signal that can be maintained for up to an hour or more.Although less sensitive than flash reactions, glow reactions can allowtime for additional sample operations (e.g., mixing, transporting, orthe like) to be completed before the signal is detected.

In some embodiments, a reagent module can be configured to deliver asubstrate into a reaction chamber in a manner that enhances themeasurement of the light signal. More particularly, in some embodiments,a reagent module can be configured to deliver a substrate in a mannerthat allows the substrate to sufficiently mix with the sample, whilealso minimizing aeration of the sample, the production of bubbles,excessive splashing, or the like, all of which can be detrimental to theoptical detection to be completed within seconds after delivering thesubstrate. For example, in some embodiments, a reagent module can definea fluidic pathway that is angled with respect to a longitudinal axis ofthe reaction chamber, such that the reagent and/or substrate isdelivered to a sidewall of the reaction chamber and then into the samplesolution. In other embodiments, a reagent module can define a fluidicpathway that is substantially parallel with respect to a longitudinalaxis of the reaction chamber, but that includes an exit openingpositioned such that the reagent and/or substrate is delivered to asidewall of the reaction chamber. In yet other embodiments, a reagentmodule can define a fluidic pathway that has a curved, arced and/orhelical shape. In yet other embodiments, a reagent module can define afluidic pathway that includes grooves, ribs, slots, or any otherflow-adjusting features, to maximize mixing and/or minimize aeration.

As another example, FIGS. 26-28 show a container assembly 4700 thatinclude a reaction chamber 4732 and a reagent module 4740. The containerassembly 4700 can be used and/or manipulated by any instrument describedherein, e.g., instrument 1100, 11000, and/or any components describedherein. The container assembly 4700 can also be used to perform anymethods described herein, e.g., methods 200 and/or 300.

The reagent module 4740 includes a housing 4741, a first actuator 4750,a second actuator 4760, a first delivery member 4772 a and a seconddelivery member 4772 b. As shown in the side cross-section view of FIG.28, the housing 4741 defines a first reagent volume 4742 and a secondreagent volume 4744. The housing 4741 can also include a deliveryportion 4770. The first reagent volume 4742 can include a first reagentcontainer 4780 a that contains a first reagent (e.g., transductionparticle). The second reagent volume 4744 can include a second reagentcontainer 4780 b that contains a second reagent (e.g., substrate). Thehousing 4741 of the container assembly 4700 can be substantially similarto the housing 3741 of the container assembly 3700 described before, andis therefore not described further in detail herein. The first reagentcontainers 4780 a and the second reagent container 4780 b are alsosubstantially similar to the first reagent container 3780 a and thesecond reagent container 3780 b, respectively, of container assembly3700 and are therefore not described in detail herein.

The first actuator 4750 includes an engagement portion 4752 and aplunger portion 4754. The second actuator 4760 includes an engagementportion 4762 and a plunger portion 4764. The first actuator 4750 and thesecond actuator 4760 are substantially similar to the first actuator3750 and second actuator 3760, respectively, of container assembly 3700as described before in structure and function, and are therefore notdescribed in further herein.

As shown in the bottom view of the reagent module 4740 in FIG. 27 andthe side cross-section of the container assembly 4700 in FIG. 28, thedelivery portion 4770 of the housing 4741 includes a first deliverymember 4772 a that provides a conduit for fluid communication of areagent, e.g., transduction particle, from the first reagent volume 4742to the reaction chamber 4732 through a first outlet 4774 a. The deliveryportion 4770 of the housing 4741 includes a second delivery member 4772b that provides a conduit for fluid communication of a reagent, e.g.,substrate, from the second reagent volume 4742 to the reaction chamber4732 through a second outlet 4774 a.

As shown in FIG. 28, the first delivery member 4772 a includes a firstportion 4775 a and a second portion 4776 a. The first portion 4775 a isat least partially disposed inside the first reagent volume 4742. Thesecond portion 4776 a is at least partially disposed inside the reactionchamber 4732. The first portion 4775 a defines a longitudinal axis thatis substantially parallel to a longitudinal axis defined by the reagentmodule 4740 and/or the reaction chamber 4732. The second portion 4776 ais angularly offset from a centerline of the first portion 4775 a suchthat the outlet 4774 a points towards a sidewall of the reaction chamber4732. Similarly stated, the second portion 4776 a is nonparallel to alongitudinal axis defined by the reagent module 4740 and/or the reactionchamber 4732. In some embodiments, the angle can be between thelongitudinal axis and the second portion 4776 a can be about 15-45degrees, inclusive of all angles therebetween. In such embodiments, thereagent and/or transduction particles conveyed from the first reagentvolume 4742 into the reaction chamber 4732 does not impinge directlyupon a surface of the sample, but instead is propelled onto or along thesidewall of the reaction chamber, such that it can flow at a controlledspeed into the sample solution.

As shown in FIG. 28, the second delivery member 4772 b includes a firstportion 4775 b and a second portion 4776 b. The first portion 4775 b isat least partially disposed inside the second reagent volume 4744. Thesecond portion 4776 b is at least partially disposed inside the reactionchamber 4732. The first portion 4775 b defines a longitudinal axis thatis substantially parallel to a longitudinal axis defined by the reagentmodule 4740 and/or the reaction chamber 4732. The second portion 4776 bis angularly offset from a centerline of the first portion 4775 b, suchthat the outlet 4774 b points towards a sidewall of the reaction chamber4732. Similarly stated, the second portion 4776 b is nonparallel to alongitudinal axis defined by the reagent module 4740 and/or the reactionchamber 4732. In some embodiments, the angle can be between thelongitudinal axis and the second portion 4776 b can be about 15-45degrees, inclusive of all angles therebetween. In such embodiments, thereagent and/or substrate conveyed from the second reagent volume 4744into the reaction chamber 4732 does not impinge directly upon a surfaceof the sample, but instead is propelled onto or along the sidewall ofthe reaction chamber, wherein it can flow at a controlled speed into thesample solution.

The first portion 4775 a, 4775 b of each of the delivery members 4772 a,4772 b includes a puncturer 4792 a, 4792 b (respectively) at an endportion thereof. In this manner, the puncturer 4792 a protrudes into thefirst reagent volume 4742, and the puncturer 4792 b protrudes into thesecond reagent volume 4744. In particular, the end of the fluidicpathways delivery members can be tapered or chamfered to produce a sharpedge that serves as the puncturers. The puncturer 4792 a and thepuncturer 4792 b can be used to puncture the frangible portion 4784 aand the frangible portion 4784 b, respectively, of the reagentcontainers to release the reagents, transduction particles or othersubstances contained therein. Although the delivery member 4772 a andthe deliver member 4772 b are shown as being constructed separately fromthe housing 4741, in some embodiments, a delivery member can beintegrally formed with the delivery portion 4770, e.g., manufactured ina single manufacturing step. In some embodiments, the delivery member4772 a and/or the delivery member 4772 b can be manufactured separately,and then disposed in cavity 4778 a and cavity 4778 b, respectively ofthe delivery portion 4770.

In some embodiments, the reagent module of the container can include ordefine fluidic pathways configured to communicate the reagents directlyinto the sample solution, and having an exit point at any suitabledistance within the container. For example, FIG. 29 shows a sidecross-section view of a container assembly 5700 according to anembodiment. The container assembly 5700 includes a reaction chamber 5732and a reagent module 5740. The reagent module includes a housing 5741that defines a first reagent volume 5742 and a second reagent volume5744. The housing 5741 contains a first actuator 5750 and a secondactuator 5760. The housing 5741 also includes a delivery portion 5770.The container assembly 5700 can be used and/or manipulated by anyinstrument described herein, e.g., instrument 1100, 11000 and/or anycomponents described herein. The container assembly 5700 can also beused to perform any methods described herein, e.g., methods 200 and/or300.

The first reagent volume 5742 includes a first reagent container 5780 athat can contain a first reagent (e.g., transduction particle of thetypes shown and described herein). The second reagent volume 5744includes a second reagent container 5780 b that can contain a secondreagent (e.g., substrate of the types shown and described herein). Thehousing 5741 of the container assembly 5700 can be substantially similarto the housing 3741 of the container assembly 3700, and is therefore notdescribed further in detail. The reagent containers 5780 a, 5780 b aresubstantially similar to the reagent containers 3780, 3780 b ofcontainer assembly 3700 and are therefore not described in detailherein. The first actuator 5750 includes an engagement portion and aplunger portion. The second actuator 5760 includes an engagement portionand a plunger portion. The first actuator 5750 and the second actuator5760 are substantially similar to the first actuator 3750 and secondactuator 3760 of container assembly 3700, and are therefore notdescribed in further herein.

The delivery portion 5770 of the housing 5741 defines a first fluidicpathway 5772 a that provides a conduit for fluid communication of afirst reagent, e.g., transduction particles, from the first reagentvolume 5742 to the reaction chamber 5732 through a first outlet 5774 a.The delivery portion 5770 of the housing 5741 also includes a secondfluidic pathway 5772 b that provides a conduit for fluid communicationof a second reagent, e.g., substrate, from the second reagent volume5744 to the reaction chamber 5732 through a second outlet 5774 b.

As shown, the fluidic pathways 5772 a, 5772 b define a longitudinal axisthat is parallel to the longitudinal axis defined by the reactionchamber 5732. This arrangement can allow the reagents to flow from thefirst reagent volume 5742 and the second reagent volume 5744 through theoutlet 5774 a and the outlet 577 b, respectively, and straight into asample solution disposed in the reaction chamber 5732. In someembodiments, a diameter of the fluidic pathway 5772 a and/or the fluidicpathway 5772 b at the respective outlet 5774 a and 5774 b can be smallerthan a diameter at the interface of the fluidic pathway 5772 a and/orthe fluidic pathway 5772 b and the first reagent volume 5742 and thesecond reagent volume 5744, respectively. In this manner, the fluidicpathways 5772 a, 5772 b perform substantially as nozzles to acceleratethe flow of the transduction particles, reagents or the like. In someembodiments, the cross-sections can be configured such that the reagentsare expelled from the outlet 5774 a and/or the outlet 5774 b atpredefined flow rate, e.g., 1 ml/sec, 2 ml/sec, 3 ml/sec, 4 ml/sec, 5ml/sec, or any other suitable flow rate, for example, to ensure rapidand complete mixing and/or minimize aeration. In some embodiments, thefluidic pathway 5772 a and/or the fluidic pathway 5772 b can beconfigured such that the outlet 5774 a and/or the outlet 5774 b aredisposed beneath a surface of the sample within the reaction chamber5732.

In some embodiments, the housing 5741 can include a series of puncturers5792 a, 5792 b located at a base of the first reagent volume 5742 andthe second reagent volume 5744, respectively. The series of puncturescan be configured to rupture the frangible portion 5784 a, 5784 b of thereagent container 5780 a, 5780 b at multiple locations, for example, toensure efficient expulsion of the reagents contained therein.

In some embodiments, a reagent module of a container can include asingle actuator and a single reagent volume. FIG. 30-32 shows a sidecross-section view of a container assembly 6700 according to anembodiment, in a first configuration, a second configuration and a thirdconfiguration, respectively. The container assembly 6700 includes areaction chamber 6732 reversibly coupleable to a reagent module 6740.The reagent module 6740 includes a housing 6741, that defines a reagentvolume 6742 that contains a first reagent container 6780 a and a secondreagent container 6780 b. The housing also includes an actuator 6750disposed in the reagent volume 6742, and a delivery portion 6770. Thecontainer assembly 6700 can be used and/or manipulated by any instrumentdescribed herein, e.g., instrument 1100, 11000 and/or any componentsdescribed herein. The container assembly 6700 can also be used toperform any methods described herein, e.g., methods 200 and/or 300.

In some embodiments, the actuator 6750 can include an engagement portion6752 and a plunger portion 6754. The engagement portion 6752 of theactuator 6750 can be configured to move the plunger portion 6754 withinthe reagent volume 6742. In some embodiments, the plunger portion 6754of the actuator 6750 includes a fluid-tight seal 6769 to fluidicallyisolate the reagent volume 6742 from a volume outside of the housing6741. In some embodiments, the seal 6769 can be, for example, a gasket,an o-ring, a rubber seal, or any suitable seal.

In some embodiments, the reagent containers 6780 a, 6780 b can bedisposed in the reagent volume 6742, such that the first reagentcontainer 6780 a is proximal to the delivery portion 6770 of the housing6741. In particular, the first reagent container 6780 a is disposedbetween the delivery portion 6770 and the second reagent container 6780b. The first reagent container can contain a first reagent, e.g., thetransduction particle. The second reagent container 6780 b can bedisposed on top of the first reagent container 6780 a, such that thefrangible portion 6784 b of the second reagent container 6780 b abutsthe bottom portion 6788 a of the first reagent container 6780 a, and thebottom portion 6788 b of the second reagent container 6780 b abuts theplunger portion 6754 of the actuator 6750. The second reagent container6780 b can contain a second reagent, e.g., a substrate such astridecanal. The reagent volume 6742 can also include a series ofpuncturers 6792 disposed in the reagent volume 6742, that are configuredto puncture the frangible portion 6784 a of the reagent container 6780a, and the frangible portion 6784 b of the reagent container 6780 b.

The delivery portion 6770 defines a fluidic pathway 6772 that can definea longitudinal axis that is parallel to a longitudinal axis defined bythe reaction chamber 6732. In some embodiments, a diameter of thefluidic pathway 6772 at the outlet 6774 can be smaller than a diameterof the fluidic pathway 6772 at the interface of the reagent volume 6742,such that the fluidic pathway 6772 substantially resembles and/orperforms as a nozzle. In some embodiments, the fluidic pathways can beconfigured such that the reagents are expelled from the outlet 6774 atpredefined flow rate, e.g., 1 ml/sec, 2 ml/sec, 3 ml/sec, 4 ml/sec, 5ml/sec, or any other suitable flow rate, for example, to ensure rapidand complete mixing and/or minimize aeration.

In operation, any suitable instrument can manipulate the engagementportion 6752 of the actuator 6750, such that the plunger portion 6754 isdisplaced from a first position as shown in the first configuration(FIG. 30) to a second position as shown in the second configuration(FIG. 31) within the reagent volume 6742. The plunger portion 6754applies a force on the bottom portion 6788 b of the second reagentcontainer 6780 b, displacing the second reagent container 6780 b from afirst position to a second position. The second reagent container 6780 bcommunicates the pressure applied by the plunger portion 6754 to thebottom portion 6788 a of the first reagent container 6780 a, through thefrangible portion 6784 b. The force causes the frangible portion 6784 aof the first reagent container 6780 a to press against the series ofpuncturers 6792, such that the frangible portion 6784 a ruptures and thereagent contained therein is communicated through the outlet 6774 of thefluidic pathways 6772 into the reaction chamber 6732.

In the third configuration (FIG. 31), the engagement portion 6752 of theactuator is manipulated further such that the plunger portion 6754 isdisplaced from the second position to a third position within thereagent volume 6742. Displacement of the plunger portion 6754 to thethird position also displaces the second reagent container 6780 b fromthe second position to a third position. In this configuration, thefirst reagent container 6780 a is emptied of the contents containedtherein and is in a collapsed state such that the puncturer 6792penetrates through the bottom portion 6788 a of the first reagentcontainer 6780 a and ruptures the frangible portion 6784 b of the secondreagent container 6780 b. The second reagent container 6780 b thereforeis placed in fluid communication with the fluidic pathway 6772 andcommunicates the reagents container therein, e.g., substrate, throughthe outlet 6774 and into the reaction chamber 6732.

In some embodiments, a container can include a single actuator withstaged actuation for delivery of a first reagent (e.g., biologic orabiologic vectors, transduction particles and/or engineered viralvector) and a second reagent (e.g., a substrate) at different stages ofactuation. FIG. 33 shows an exploded view of a container assembly 7700that includes a reaction chamber 7732 and a reagent module 7740. FIGS.34-36 show the container assembly 7700 in a first configuration, asecond configuration and a third configuration, respectively. Thereaction chamber 7732 can be removably coupleable to the reagent module7740. The container assembly 7700 can be used with and/or manipulated byany of the instruments described herein, e.g., instrument 1100, 11000and/or any of the components described herein. The container assembly7700 can also be used to perform any of the methods described herein,e.g., methods 200 and 300.

The reaction chamber 7732 can be formed from a light weight, rigid andinert material, e.g., plastics. At least a portion of the reactionchamber 7732 can be partially transparent, e.g., to allow detectionand/or viewing of the internal volume of the reaction chamber 7732, forexample, to detect a luminescence reaction occurring therein. In someembodiments, the reaction chamber 7732 can be shaped as a cylinder witha rounded bottom, or flat base. In some embodiments, the reactionchamber 7732 can have any other suitable shape, e.g., square,rectangular, oval, polygonal, etc. In some embodiments, the reactionchamber 7732 can have a diameter of 12 mm and a height of 75 mm. In someembodiments, the container assembly 7700 can include one or moresolutions/reagents (e.g., bacterial nutrient solution, buffers,surfactants, transduction particle, and/or antibiotics), predisposedwithin the reaction chamber 7732. The reaction chamber 7732 can includethreads 7745 for removably coupling with the reagent module 7740. Insome embodiments, the reaction chamber 7732 can be removably coupled tothe reagent module 7740 via a snap-fit, friction fit, or any othersuitable mechanism.

As shown in FIG. 33, the reagent module 7740 can include a housing 7741.The housing 7741 can be formed from a lightweight and rigid material,e.g., injection molded plastic. The housing 7741 includes a firstcompression support 7748 a and a second compression support 7748 b. Thecompression supports 7748 a, 7748 b are configured to fixedly orremovably mount a first reagent container 7780 a and a second reagentcontainer 7780 b, respectively, and provide a rigid support duringcompression to the first reagent container 7780 a and the second reagentcontainer 7780 b, as described further below. The compression supports7748 a, 7748 b can include features for mounting the reagent containers7780 a, 7780 b thereto. Such mounting features can include, for example,notches, grooves, detents, indent, slot, and/or an adhesive surface. Asurface of the each of the compression supports 7748 a, 7748 b on whichthe respective reagent container 7780 a, 7780 b are mounted, are angledwith respect to a longitudinal axis defined by the housing 7741 and/orthe reaction chamber 7732. The angled surface can be beneficial, forexample, to allow smooth (e.g., even and/or controlled) flow of thereagent and/or substrate and with low dead volume, from the reagentcontainers 7780 a, 7780 b into the reaction chamber 7732.

The reagent module 7740 includes an actuator 7750, that is configured toslide within an internal volume defined by the housing 7741. Theactuator 7750 is configured such that a sidewall of the actuator 7750and a sidewall of the housing 7741 form a fluid-tight seal. Thus, inuse, the actuator 7750 can be displaced within the housing 7741 whilemaintaining a substantially fluid-tight seal. As shown in FIG. 34, thehousing 7741 and the actuator 7750 can be configured to define a firstreagent volume 7742 and a second reagent volume 7744 that are separatedat least partially by a sidewall 7746 (FIG. 34-36), and at leastpartially by a sidewall of the compression supports 7748 a, 7748 b. Theactuator includes an engagement portion 7752 configured to bemanipulated by an instrument, e.g., instrument 1100 or any otherinstrument shown and described herein.

The actuator 7750 includes a first compression member 7754 that can beshaped to resemble, for example, an angled clip. The first compressionmember 7754 can be a separate component that can be formed from a rigidmaterial, e.g., aluminum, steel, stainless steel, or plastics. The firstcompression member 7754 has an engagement portion 7755 and a compressionportion 7756, that is inclined at an angle away from the longitudinalaxis defined by the housing 7741. In particular, the angle issubstantially similar to the angle defined by the angled surface of thefirst compression support 7748 a. The first compression member 7754 alsoincludes a sliding portion 7757 that abuts the sidewall 7746 of theactuator 7750 and is configured to slide in a space 7747 between thesidewall 7746 and the first compression support 7748 a as describedfurther below. The engagement portion 7755 of the first compressionmember 7754 can include an engagement spring 7758 coupled to theengagement portion 7755. The engagement spring 7758 can be a compressionspring, for example, a helical spring, coil spring, Belleville spring,or tapered spring and can include washers (not shown) for mounting onthe engagement portion 7755. An end of the engagement spring 7758 distalto the first compression member 7754 can be coupled to the actuator7750, for example, mounted on a pin, mandrel, or the likes, furtherconfigured such that engaging the actuator 7750, engages the engagementspring 7758 and the first compression member 7754.

The actuator 7750 can also include a second compression member 7760 thatcan be an integral part of the actuator 7750, for example, formed in thesame injection molded process. The second compression member 7760 can beshaped to resemble an inclined plane that is angled away from thelongitudinal axis defined by the housing 7741. The angle can besubstantially similar to the angle defined by the inclined surface ofthe second compression support 7748 b. In some embodiments, the firstcompression member 7754 and/or the second compression member 7760 caninclude one or a series of puncturers, e.g., thorns, barbs, pins, or anyother suitable puncturing member to puncture a frangible portion of thereagent containers 7780 a, 7780 b, as described herein.

The housing 7741 can also include a first fluidic outlet 7774 a and asecond fluid outlet 7774 b for communicating reagents from the firstreagent volume 7742, e.g., the biologic or abiologic vectors,transduction particle and/or engineered viral vector, and the secondreagent volume 7744 e.g., substrate, into the reaction chamber 7732. Thefluidic outlets 7774 a, 7774 b can be an opening and/or space betweenthe compression supports 7748 a, 7748 b and a sidewall of the housing7741. In some embodiments, the housing 7741 can include fluidicpathways, e.g., nozzles, angled nozzles, tubes, and/or any othersuitable fluid conduit, e.g., to facilitate rapid mixing and/or minimizeaeration, as described herein.

The first reagent container 7780 a and the second reagent container 7780b can be disposed on the first compression support 7748 a and the secondcompression support 7748 b, respectively as described before. Thereagent containers 7780 a, 7780 b can each include a sidewall 7782 a,7782 b and a frangible member 7784 a, 7784 b that collectively define aninternal volume. The internal volume of the reagent containers can becompletely or partially filled with a reagent, e.g., the first reagentcontainer 7780 a can contain transduction particles (e.g., transductionparticle 160 or any other transduction particles described herein), thatcan include an engineered nucleic acid (e.g., engineered nucleic acid170) formulated to cause the target cell (e.g., bacteria) to produce aplurality or reporter molecules (e.g., luciferase). The second reagentcontainer 7780 b can contain a substrate, e.g., tridecanal, that caninteract with the reporter molecule, e.g., luciferase, to trigger,catalyze, produce and/or enhance the production of a measurable signal,e.g., via a luminescence reaction.

The reagent containers 7780 a/b can be constructed from materials thatare substantially impermeable to and/or substantially chemically inertfrom the substance contained therein, e.g., transduction particle,substrate, antibiotics, buffers, surfactants, or any other reagent thatcan be required for the detection assay. In this manner, the reagentscan be stored in the reagent containers 7780 a/b for extended periods oftime. For example, the reagent containers 7780 a/b side wall 7782 a/bcan be formed from a flexible and inert material, e.g., blister plastic,aluminum foil, or any other suitable material. Moreover, in someembodiments, the frangible member 7784 a/b can be constructed from amaterial having certain temperature characteristics such that thedesired properties and integrity of the frangible member 7784 a/b aremaintained over a certain temperature. For example, in some embodiments,it can be desirable to store the reagent container 7780 a/b containingreagent or substrate in a refrigerated condition or it can be desirableto manufacture the reagent container 7780 a/b by thermally laminatingthe frangible member 7784 a/b. In such embodiments, the frangible member7784 a/b can be selected such that the refrigeration condition and/orthermal lamination condition do not substantially degrade the desiredproperties and integrity of the frangible member 7784 a/b for theintended application. In some embodiments, the frangible member 7784 a/bcan be constructed from a polymer film, such as any form ofpolypropylene. In some embodiments, the frangible member 7784 a/b can beconstructed from bi-axially oriented polypropylene (BOP). In someembodiments, the frangible member 7784 a/b can be constructed fromaluminum. The frangible portions 7784 a/b of the reagent containers canbe configured to rupture when compressed, e.g., by the compressionmembers 7754/7760 of the actuator 7750 as described further below, andrelease the reagent contained therein.

As shown in FIG. 34 the container assembly 7700 can initially bemaintained in a first configuration, in which the reagent module 7740 iscoupled to the reaction chamber 7732, and the actuator 7750 is in afirst position. The first compression member 7754 is in a firstposition, in which the engagement portion 7756 is in contact with thefrangible portion 7784 a of the first reagent container 7780 a but notapplying any compressive force, and the engagement spring 7758 is fullyuncompressed. The second compression member 7760 is also in a firstposition wherein the second compression member is not contacting thefrangible portion 7784 b of the second reagent container 7780 b.

In the second configuration (FIG. 35), the engagement portion 7752 ofthe first actuator 7750 is manipulated to displace the actuator 7750within the housing 7741 from the first position, to a second position.The displacement of the actuator 7750 urges the engagement spring 7758to compress and exert a force on the first compression member 7754. Theforce displaces the first compression member 7754, from the firstposition to a second position as shown in FIG. 35, wherein the slidingportion 7757 of the first compression member 7754 slides with thesidewall 7746 into the opening 7747 between the first and secondcompression supports 7748 a, 7748 b. The engagement portion 7756 of thefirst compression member 7754 exerts a compressive force on thefrangible portion 7784 a of the first reagent container 7780 a, suchthat the frangible portion 7784 a ruptures, releasing its contents(e.g., transduction particles) into the first reagent volume 7742. Thereagent then flows through the first outlet 7774 a and into a solutionin the reaction chamber 7732, e.g., a patient sample containing targetcell. The second compression member 7760 is also displaced from thefirst position to a second position, wherein it is near the frangibleportion 7784 b of the second reagent container 7780 b and/or contactingthe frangible portion 7784 b without rupturing it.

In the third configuration (FIG. 36), the engagement portion 7752 of theactuator 7750 is manipulated to displace within the housing 7741 fromthe second position to a third position. The displacement of theactuator 7750 further urges the engagement spring 7758 to compress andexert a force on the first compression member 7754. In this positionfurther displacement of the first compression member 7754 is preventedby the first compression support 7748 a, and the engagement spring 7758is substantially compressed. The second compression member 7760 is alsodisplaced from the second position to a third position, wherein thesecond compression member 7760 exerts a compressive force on thefrangible portion 7784 b of the second reagent container 7780 b. Thiscauses the frangible portion 7784 b to rupture and release its contents,e.g., substrate into the second reagent volume 7744. The reagent thenflows through the second outlet 7774 b and into a solution in thereaction chamber 7732, e.g., a sample containing target cell andreporter molecules produced by the target cells.

As described above, in some embodiments, a delivery portion can beconfigured to deliver one or more reagents into a reaction chamber in amanner that promotes mixing, that minimizes aeration, overspray and/orundesirable turbulence. For example, in some embodiments, a reagentmodule of a container can include a delivery portion that is inclinedand/or angularly offset with respect to a longitudinal axis of thereagent module and/or the reaction chamber. Such an arrangement candirect a flow of reagent (e.g., transduction particles, substrate or thelike) in a manner that improves mixing, detection or the like. Inparticular, FIGS. 37-38 show a side cross-sectional schematic view of acontainer assembly 8700 according to an embodiment, in a firstconfiguration and a second configuration, respectively. The containerassembly 8700 can be used with and/or manipulated by any of theinstruments described herein, e.g., instrument 1100, 11000 and/or any ofthe components described herein. The container assembly 8700 can also beused to perform any of the methods described herein, e.g., methods 150,200 and 300.

The container assembly 8700 includes a reaction chamber 8732 that isreversibly coupleable to a reagent module 8740. The reaction chamber8732 can have a sample S disposed therein, e.g., a sample solutionincluding a patient sample, target cell, transduction particles, and/orreporter molecules. In use, the container assembly 8700 can beoperatively coupled to a detector 1200 such that reaction chamber 8732is in communication (e.g., optical communication) with the detector1200. Although the embodiments described herein have been shown as beingoptically coupled to the detector 1200, any other detector describedherein can be used.

The reagent module 8740 includes a housing 8741, a delivery member 8770and an actuator 8750. The housing 8741 defines a reagent volume 8742,which can contain a reagent disposed therein. The reagent can be anysuitable reagent, such as any of the substrates described herein.

The actuator 8750 includes a plunger portion 8754 configured to be influid communication with the reagent disposed in the reagent volume8742. The actuator 8750 can be configured to convey the reagent from thereagent volume 8742 to the reaction chamber 8732 through the deliverymember 8770 (e.g., via a fluidic pathway 8772). The delivery member 8770includes a first portion 8774 that is disposed substantially inside thereagent volume 8742 and proximal to the plunger. The delivery member8770 also includes a second portion 8776 disposed substantially insidethe reaction chamber 8732, when the reaction chamber 8732 is coupled tothe reagent module 8740.

The delivery member 8770 is angularly offset with respect to alongitudinal axis of the reagent module 8740 and/or the reaction chamber8732. More particularly, as shown in FIG. 37, the centerline of thefluidic pathway that defines the axis of the delivery member 8770 isoriented at an angle θ away from the longitudinal axis. In someembodiments, the angle θ can be about 30 degrees. In some embodiment,the angle θ can be between about 15-45 degrees.

In operation, the actuator 8750 can be manipulated by applying a forceon the actuator 8750 as shown by the arrow AA in FIG. 38, e.g., using amanipulation mechanism of the instrument 1100. This urges the plungerportion 8754 to be displaced within the reagent volume 8742 from a firstposition, as shown in the first configuration (FIG. 37), to a secondposition as shown in the second configuration (FIG. 38). The plungerportion 8754 conveys and/or expels the reagent (e.g., substrate)contained in the reagent volume 8742 through the fluidic pathway 8772.Similarly stated, the plunger portion 8754 is moved within the reagentvolume along the longitudinal axis to produce a flow of a reagent fromthe reagent volume via the fluidic pathway 8772.

As shown in the FIG. 38, the inclined orientation of the deliveryportion 8770 causes the expelled reagent to follow an inclined path asshown by arrow BB (FIG. 38), such that the reagent stream impinges on asidewall of the reaction chamber 8732. The reagent stream then flowsdown the sidewall of the reaction chamber 8732 as shown by arrow CC(FIG. 38) and mixes with the sample S at a lower fluid velocity, therebyminimizing and/or eliminating aeration and/or the production of bubbleswithin the sample. Minimizing aeration can, enable mixing of the reagentwith the sample S and increase the quality of the signal that isdetected by the detector 1200. For example, in some embodiments, thecontainer assembly 8700 can be used in conjunction with a reportersystem and reagent (e.g., substrate) that are collectively formulated toproduce a flash reaction in response to the addition of the substrate tothe sample within which reporter molecules have been expressed. In suchembodiments, the arrangement of the delivery member 8770 can allow thesubstrate to sufficiently mix with the sample, while also minimizingaeration of the sample, the production of bubbles, excessive splashing,or the like, all of which can be detrimental to the optical detection tobe completed a short time period (e.g., within seconds) after deliveringthe substrate.

Although the reagent module 8740 is shown and described above asincluding a single delivery member that directs the flow of a reagentalong a portion of a side wall of the reaction chamber 8732, in otherembodiments, a reagent module 8740 can include multiple differentdelivery members and/or a delivery member with multiple exit points tofacilitate rapid delivery of the reagent therein, while also minimizingaeration, the production of bubbles or the like. In some embodiments, areagent module of a container can include a delivery member that isconfigured to produce an annular flow of a reagent disposed in thereagent module, such that the reagent flows substantiallycircumferentially about the sidewall of the reaction chamber. Forexample, FIGS. 39-40 show a side cross-sectional view of a containerassembly 9700 according to an embodiment, in a first configuration and asecond configuration. The container assembly 9700 includes a reactionchamber 9732 reversibly coupleable to a reagent module 9740. Thecontainer assembly 9700 is coupled to a detector 1200 such that reactionchamber 9732 is in optical communication with the detector 1200. Thecontainer assembly 9700 can be used and/or manipulated by any instrumentdescribed herein, e.g., instrument 1100, 11000 and/or any componentsdescribed herein. The container assembly 9700 can also be used toperform any methods described herein, e.g., methods 150, 200 and/or 300.In some embodiments, the container assembly 9700 can be used to detect asignal produced by a flash luminescence reaction. While the embodimentsdescribed herein have been shown optically coupled to the detector 1200,any other detector described herein can be used.

The reaction chamber 9732 can have a sample S disposed therein, e.g., asample solution including a patient sample, target cell, transductionparticles, and/or reporter molecules. The reaction chamber 9732 can besimilar to any of the reaction chambers described herein, and istherefore not described in detail.

The reagent module 9740 includes a housing 9741 defining a reagentvolume 9742, which can have a reagent disposed therein, e.g., asubstrate. The housing 9741 also includes an actuator 9750 disposed inthe reagent volume 9742. The reagent module 9740 includes a frangibleportion 9784 and a delivery member 9770.

The actuator 9750 includes a plunger portion 9754 in fluid communicationwith the reagent disposed in the reagent volume 9742. The plungerportion 9754 is configured to move within the housing 9741 to convey thereagent from the reagent volume 9742 to the reaction chamber 9732through the frangible portion 9784.

The delivery member 9770 is configured to define a puncturing portion9772 and a rounded and/or curved sidewall 9774. In some embodiments, thesidewalls 9774 can be tapered, contoured, and/or include gradations.Although the delivery portion 9770 is shown as being locatedsubstantially inside the reaction chamber 9732, in other embodiments, asubstantial portion of the delivery member 9770 can be disposed withinand/or coupled to the reagent module 9740. The delivery member 9770 canbe an integral part of either the reaction chamber 9732 or the reagentmodule 9740.

As shown, when the reagent module 9740 is in the first configuration(FIG. 39), the puncturing portion 9772 is in contact with the frangibleportion 9784 but is not applying any puncturing force on the frangiblemember 9784. Accordingly, the reagent within the reagent volume 9742 ismaintained in fluidic isolation from the reaction chamber 9732. When thereagent module 9740 is moved to the second configuration (FIG. 40), aforce is applied on the actuator 9750 in a direction as indicated byarrow DD. This causes the plunger portion 9754 to displace from thefirst position to a second position. The displacement of the plungerportion 9754 exerts a force on the frangible member 9784 (via thereagent). This causes the frangible member to press against thepuncturing portion 9772 of the delivery portion and puncture (FIG. 40),thereby puncturing the frangible portion 9784. The contour and/or shapeof the delivery member 9770 produces an annular flow of the reagentaround the entire sidewall 9774 of the reaction chamber 9732, as shownby arrow EE. The reagent stream can then flow down along a sidewall ofthe reaction chamber 9732, thereby minimizing and/or eliminatingaeration, the production of bubbles or the like.

In some embodiments, a reagent module of a container can include adelivery portion or delivery member that is curved. FIG. 41 shows a sidecross-section of a container assembly 10700 according to an embodiment.The container assembly 10700 includes a reaction chamber 10732reversibly coupleable to a reagent module 10740. The reagent module10740 includes a housing 10741 defining a reagent volume 10742, whichcan have a reagent disposed therein, e.g., a substrate. The housing alsoincludes an actuator 10750 disposed in the reagent volume 10742. Thecontainer assembly 10700 includes a delivery member 10770. The containerassembly 10700 can be used and/or manipulated by any instrumentdescribed herein, e.g., instrument 1100, 11000 and/or any componentsdescribed herein. The container assembly 10700 can also be used toperform any methods described herein, e.g., methods 150, 200 and/or 300.In some embodiments, the container assembly 10700 can be used to detecta signal produced by a flash luminescence reaction.

The actuator 10750 includes a plunger portion in fluid communicationwith the reagent, e.g., a substrate, disposed in the reagent volume10742. The plunger portion 10754 is configured to displace within thereagent volume 10742 to convey the reagent from the reagent volume 10742to the reaction chamber 10732 through the delivery portion 10770

The delivery member 10770 is shaped such that it defines a curvedfluidic pathway 10772. The curved fluidic pathway 10772 is configuredsuch that the reagent is dispensed from the fluidic pathway in aswirling motion. The swirling motion can, for example, create turbulencein the reagent flow that can, for example, enhance mixing of the reagentwith a sample contained in the reaction chamber 10732. In someembodiments, the curved fluidic pathways 10772 can cause the reagent toimpinge on a sidewall of the reaction chamber. The reagent can then flowalong the sidewall of the container to reach the sample at a lowervelocity, for example, to minimize aeration.

The container assembly 4700 or any of the container assemblies describedherein, can be operably coupled to a detector of any suitable type todetect a signal produced and/or enhanced by the interaction of areporter molecule (e.g., luciferase) with a substrate (e.g.,tridecanal). As described above, in some embodiments, methods ofdetection can include detection of a signal produced by a flashluminescence reaction. Such methods can include, for example, directinga flow of a reagent (e.g., substrate) in a manner to enhance theaccuracy with which the signal is read. Similarly stated, such methodscan include, for example, directing a flow of a reagent (e.g.,substrate) in a manner to minimize undesirable turbulence, aeration, theproduction of bubbles or the like. For example, FIG. 42 shows a method400 for detecting target cells using a container assembly and adetector. The method 400 can be used with any of the containerassemblies described herein. The detector can be the detector 1200, thedetector assembly 11200, or any other detector described herein. Areaction chamber of the container assembly can include a sample disposedtherein. The sample can be, for example, a sample solution containing apatient sample target cell (e.g., a nasal swab potentially containingMRSA), biologic or abiologic vector such as a transduction particle(e.g., any of the transduction particles described herein), and a seriesof reporter molecules produced in accordance with any of the reportersystems disclosed herein.

The method includes operably coupling the reaction chamber of thecontainer to a detector, 402. A reagent is then communicated into thereaction chamber using a delivery member, 404. In some embodiments, thereagent can be any suitable substrate. In some embodiments, the reagentcan include a 6-carbon aldehyde (hexanal), a 13-carbon aldehyde(tridecanal) and/or a 14-carbon aldehyde (tetradecanal), inclusive ofall the varying carbon chain length aldehydes therebetween. In someembodiments, the reagent can be formulated to include Tween 20 or anyother surfactant, tridecanal or other aldehydes, and adjusted to aparticular pH. The reagent can be disposed in a reagent module of thecontainer, e.g., a reagent module 4740 of the container assembly 4700,or any other reagent module as described herein.

The delivery member can be, for example, the delivery portion 3770, thedelivery member 4770, or any other structure and/or mechanism thatdefines a flow path through which the reagent can be conveyed. Thereagent is conveyed in a manner to flow along a surface of the reactionchamber and into the sample, 406. In this manner, as described herein,the delivery of the reagent into the sample can be performed in a mannerthat enhances the accuracy with which the signal is read. In someembodiments, the conveying can include conveying the reagent in adirection non-perpendicular to a surface of the sample. In someembodiments, the reagent is conveyed at a flow rate of at least onemilliliter per second. In some embodiments, the conveying includesmoving a plunger in a direction within a reagent volume, a first endportion of the delivery member disposed within the reagent volume and asecond end portion of the delivery member disposed within the reactionchamber. In some embodiments, the movement of the delivery memberconveys the member in an exit direction non-parallel to the direction,e.g., the delivery portion 4770.

On reaching the sample, the reagent reacts with the series of reportermolecules and produces a signal, 408. The production of the reportermolecules can be in accordance with any of the systems, compositions andmethods described herein. The signal is received via the detector, 410.The signal can be used, for example, to determine a viable cell and/orto report a gene present within a target cell in the sample. In someembodiments, the receiving of the signal is performed for less than 60seconds after the conveying of the reagent.

Any of the container assemblies described herein can be manipulated,handled and/or actuated by any suitable instrument to perform anidentification and/or detection process on a sample contained within thecontainer assembly in accordance with any of the methods disclosedherein. For example, in some embodiments, any of the containerassemblies described herein can be manipulated and/or actuated by aninstrument to perform viable cell reporting and/or gene reporting of atarget cell within a sample using biologic or abiologic vectors such astransduction particles methods and/or reporter systems of the typesshown and described herein. In this manner, a system (e.g., thecontainer or a series of container, the transduction particles, reagentsand other compositions, and an instrument) can be used for manydifferent assays, such as, for example, the rapid and/or automateddetection of MRSA, C. difficile, Vancomycin resistant Enterococci, etc.In some embodiments, an instrument can also be configured to facilitate,produce, support and/or promote a reaction in a sample contained in acontainer and or series of containers of the types shown and describedherein. Such reactions can include, for example, interaction and/ormixing of a transduction particles (e.g., any of the transductionparticles described herein) with a sample, interaction and/or mixing ofa reporter molecule (e.g., luciferase) with a substrate (e.g.,tridecanal). Such reactions can, in accordance with the methodsdescribed herein, produce a signal, e.g., via a luminescence reaction,that can be detected by components included in the instruments describedherein.

In some embodiment, a system can include an instrument including varioussubassemblies and configured to manipulate a container, actuate anactuation mechanism of a container, maintain a container and/or detect asignal produced in the container. For examples FIGS. 43-45 show aportion of an instrument 1100 in a first configuration, a secondconfiguration and a third configuration, respectively. The instrument1100 is configured to receive a container, e.g., container 11700, thatcan include a sample disposed therein, and can further be configured tomanipulate the container 11700 and detect a signal produced therein,e.g., via a luminescence reaction. The container 11732 defines a reagentvolume 11742 and a reaction volume 11732. The instrument 1100 includes ahousing 1110 that defines an internal volume that contains and/orsupports a detector 1212, a retention member 1610, an activation member1636 and an actuator 1652. While shown as receiving container 11700, theinstrument 1100 can be configured to receive any container assemblydescribed herein, and can be used to perform any methods describedherein, e.g., method 150, 200, 300 or 400.

As shown, the retention member 1610 is configured to contact a firstportion 11733 of a sample container 11700 disposed in the instrument1100 to limit movement of the container 11700. Similarly stated, theretention member 1610 is configured to contact the sample container11700 to limit movement lateral movement and/or rotation of the samplecontainer 11700 relative to portions of the instrument 1100 (e.g., thedetector 1212). In some embodiments, the retention member 1610 can beconfigured to limit periodic movement (e.g., vibration) of the samplecontainer 11700.

The activation member 1636 is coupled to the actuator 1652 and is alsomovably coupled to the retention member 1610. The activation member 1636is configured to engage a second portion 11752 of the container 11700 toconvey a reagent, e.g., substrate (e.g., tridecanal) from the reagentvolume 11742 into the reaction volume 11732. The actuator 1652 isconfigured to move the activation member 1636 between a first position(FIG. 43), a second position (FIG. 44) and a third position (FIG. 45).Similarly stated, the actuator 1652 is configured to move the activationmember 1636 relative to the instrument 1100 and/or the retention member1610.

When the activation member 1636 is in the first position, the retentionmember 1610 is spaced apart from the first portion 11733 of thecontainer 11700 and the activation member 1636 is spaced apart from thesecond portion 11752 of the container 11700. In this manner, thecontainer 11700 can be moved relative to the detector 1212 (e.g., toposition the container or the like. When the activation member 1636 isin the second position (FIG. 44) the retention member 1610 is configuredto contact the first portion 11733 of the container 11700. In thismanner, movement of the sample container 11700 relative to portions ofthe instrument 1100, such as the detector is limited. Furthermore, theactivation member 1636 remains spaced apart from the second portion11752 of the container 11700 in the second configuration. As shown inFIG. 45, when the assembly is moved to the second configuration, anengagement surface 1620 of the retention member 1610 is in contact witha corresponding surface 1644 included in the activation member 1636. Thesurface 1644 is shaped and/or configured to urge the engagement surface1612 of the retention member 1610 to contact the first portion 11733 ofthe container 11700.

More particularly, as shown in FIG. 44, when the assembly is moved tothe second configuration, the actuator 1652 moves the activation member1636 in a first direction (shown by the arrow AA) defined by thelongitudinal axis A_(L) of the container 11700. The engagement of theengagement surface 1620 of the retention member 1610 and thecorresponding surface 1644 of the activation member 1636 causes theretention member 1610 to move in a second direction, as shown by thearrow BB in FIG. 44. Similarly stated, the activation member 1636engages the retention member 1610 to move the retention member 1610 in adirection perpendicular to the longitudinal axis A_(L) and towards thecontainer 11700.

As shown in FIG. 44, in the second configuration (when the activationmember 1636 is in the second position), the container 11700 is disposedin the housing 1110 and/or in contact with a portion of the housing 1110such that the container 11700 is separated from the detector 1212 by thedistance d. The distance d defines the signal path length (e.g., opticalpath length) to be maintained during detection. Moreover, the retentionmember 1610 maintains the container 11700 in contact with a portion ofthe housing 1110 such that lateral movement, sideways wobble or verticalmotion of the container 11700 is limited and/or eliminated (see, e.g.,the arrow CC). Maintaining a consistent and repeatable signal pathlength can result in a repeatable and high quality measurement of thesignal produced in the reaction volume of the container 11700. In someembodiments, the instrument 1100 can be used in conjunction with any ofthe methods described herein to detect a signal produced by a flashluminescence reaction.

When the activation member 1636 is in the third position (correspondingto a third configuration), the activation member 1636 is engaged withthe second portion 11752 of the container 11700 to convey the reagentfrom the reagent volume 11742 to the reaction volume 11732, as shown bythe arrow DD in FIG. 45. In some embodiments, the activation member 1636can include a plunger portion configured to engage the container 11700and move within the reagent volume 11742 of the container 11700, whenthe activation member 1636 moves from the first position to the secondposition. More particularly, when moved to the third configuration (FIG.45), the actuator 1652 moves the activation member 1636 further alongthe direction shown by arrow AA, such that a plunger portion of theactivation member 1636 contacts an engagement portion 11752 of thecontainer 11700 and then moves into the reagent volume 11743. As theactivation member 1636 moves from its second position to its thirdposition (i.e., within the reagent volume 11742), it conveys the reagentcontained therein, e.g., substrate such as tridecanal, into the reactionvolume 11732. The reagent flows into the sample S and interacts with thesample S to produce a signal that can be detected by the detector 1212.For example, the reagent can be tridecanal that interacts with thereporter molecule luciferase present in the sample solution. Theinteraction produces luminescence which is detected by the detector 1212which can be, e.g., a photodetector.

When the assembly is moved from the second configuration to the thirdconfiguration, the retention member 1610 remains in contact with thefirst portion 11733 of the container 11700 in the third configuration.In this manner, the reagent, which can be, for example, a substrate, canbe added to the sample when the container is in a fixed positionrelative to the detector 1212. As discussed above, this arrangementfacilitates repeatable measurement of the signal.

In some embodiments, the instrument 1100 can also include a secondactivation member (not shown) which can be configured to engage a thirdportion of the sample container 11700, e.g., to convey a second reagente.g., transduction particle, from the second reagent volume into thereaction volume. The second activation member can be movably coupled tothe first activation member 1636, and can be configured to contact thethird portion of the container 11700 when the activation member 1636 isin the first position (i.e., the assembly is in the firstconfiguration). Thus, in such embodiments, when the assembly is in thefirst configuration the second activation member can be moved (e.g., bya second actuator) to convey a second reagent.

In some embodiments, at least a portion of the second activation membercan be movably disposed within the first activation member 1636. In someembodiments, the retention member 1610 can also be configured to rotatewhen the activation member 1636 is displaced from the first position tothe second position.

In some embodiments, a portion of an instrument 2100 can include aretention assembly that can include a series of grippers to manipulateand/or actuate a container of the types shown and described herein. Forexample, referring now to FIGS. 46-48, an instrument 2100 includes adetector 2212, a retention assembly 2610, an instrument 2100, anactivation member 2636 and an actuator 2652. The instrument 2100includes the container assembly 11700, which is described herein withreference to FIGS. 43-45. The instrument 2100 can, however, be used toreceive and/or manipulate any other container described herein, and canbe used to perform any methods described herein, e.g., methods 150, 200,300 or 400.

As shown in FIGS. 43-45, the retention assembly 2610 includes a firstgripper 2612 a and a second gripper 2612 b each configured to contact afirst portion 11733 of the container 11700 to limit movement of thecontainer 11700. In this manner the retention assembly, among otherfunctions, can facilitate repeatable detection as described before withreference to FIGS. 43-45. Each gripper is coupled to a biasing member2622, e.g., a spring, configured to exert a force on the correspondinggrippers 2612 a, 2612 b to urge the grippers towards a closedconfiguration.

The activation member 2636 is movably coupled to the retention assembly2610, and is operably coupled to the actuator 2652. The actuator 2652 isconfigured to move the activation member 2636 relative to the retentionassembly 2610 and/or the container 11700 between a first position and asecond position. The activation member includes a first surface 2642configured to contact with a surface of the retention assembly 2610 tomaintain the first gripper 2612 a and the second gripper 2612 b in anopened configuration when the activation member 2636 is in the firstposition. For example, FIG. 46 shows the instrument 2100 in the firstconfiguration such that the activation member 2636 is in the firstposition and the surface 2642 of the activation member 2636 is incontact with the retention assembly 2610, thereby maintaining grippers2612 a, 2612 b in the open configuration.

In the second position, a second surface 2644 of the activation member2636 is configured to contact the surface of the retention assembly 2610such that the biasing member 2622 urges the first gripper 2612 a and thesecond gripper 2612 b into a closed configuration. More particularly,when moving towards the second configuration (FIG. 47), the actuator2652 moves the activation member 2636 from its first position to itssecond position along a longitudinal axis B_(L) of the container 11700in the direction shown by arrow FF. This motion of the activation member2636 causes the retention assembly 2610 to be in contact with the secondsurface 2644 of the activation member 2636. In this position, thebiasing members 2622 urge the gripper 2612 a, 2612 b to move in a seconddirection as shown by the arrow EE such that the grippers 2612 a, 2612 bcontact the first portion 11733 of the container 11700. In someembodiments, the movement of the activation member 2636 from the firstposition to the second position can also cause the grippers to rotatetowards the container 11700.

The activation member 2636 is configured to engage a second portion11752 of the container 11700 and convey a reagent, e.g., substrate suchas tridecanal, from the reagent volume 11742 into the reaction chamber11732. More particularly, when moving towards the third configuration,as shown in FIG. 48, the actuator 2652 can continue to move theactivation member 2636 in the direction shown by the arrow FF, until theplunger portion 2638 of the activation member 2636 contacts a secondportion 11752 of the container 11700, and moves from a first position toa second position within the reagent volume 11742 of the container11700. The plunger portion 2638 can therefore communicate the reagentdisposed in the reagent volume e.g., substrate such as tridecanal, intothe reaction volume 11732. The substrate can react with reportermolecules, e.g., luciferase, included in the sample S disposed in thereaction volume 11732 to produce a signal, e.g., luminescence that canbe detected by the detector 2212.

As shown, the grippers 2612 a, 2612 b remain in contact with andotherwise engage, grasp, clamp or secure the container 11700 in thethird configuration. This prevents lateral movement, sideway wobble orvertical motion of the container 11700 as shown by arrow GG, andmaintains a consistent signal path length d from the detector 2212, asdescribed above. As shown, the retention and the actuation (e.g., theplunging) of the container assembly 11700 is accomplished using a singleactuator, and with the retention assembly 2610 and the activation member2636 cooperatively configured such that the reagent cannot be conveyedinto the sample container 11700 unless the sample container 11700 is inthe desired position for the detection operation.

In some embodiments, an instrument can include a detector assembly thatcan include a housing for receiving a sample container and a shutterconfigured to minimize background light, facilitate calibration of thesignal detection or the like. For example, as shown in FIGS. 49-50, aninstrument can include detector assembly 3200. The detector assembly3200 includes a housing 3202, a detector 3212 and a shutter 3230. Thedetector assembly 3200 is configured to receive a container 12700 toanalyze a signal produced therein. Such signals can be produced by anyof the methods described herein, such as, for example, resulting fromthe interaction of a substrate and a reporter molecule. In someembodiments, the signal can be produced by a flash luminescencereaction. The detector assembly 3200 can be configured to receive anyother container, e.g., container 1700 or any other container describedherein, and can be used to perform any method described herein, e.g.,method 150, 200, 300 or 400.

As shown, the housing 3202 defines a channel 3209 and a detection volume3234. The channel 3209 is configured to receive the container 12700, orany other container. The detection volume 3234 is configured to placethe channel 3209 in communication with the detector 3212. The housing3202 further includes a first seal surface 3206 and a second sealsurface 3207. In use, a first portion 12733 of the container 12700 andthe first seal surface 12733 isolate the detection volume 3234 from avolume outside the housing 3202 when a second portion 12732 of thecontainer 12700 is disposed within the detection volume 3234 (see e.g.,FIG. 50). In this manner, the first portion 12733 of the container 12700and the first seal surface 12733 can eliminate and/or limit the amountof background noise (e.g., ambient light) within the detection volume3234 when the container 12700 is positioned within the channel 3209 fordetection of the sample contained therein.

The shutter 3230 is disposed within the housing 3209. The shutter 3230is movable between a first shutter position (FIG. 49) and a secondshutter position (FIG. 50). When the shutter is in the first shutterposition (i.e., a first configuration of the assembly), the shutter 3230is in the first position such that the second seal surface 3207 of thehousing 3202 and a seal surface 3231 of the shutter are in contact andisolate the detection volume 3234 from the channel 3209. In someembodiments, the first seal surface 3206 can be a gasket. In thismanner, when in the first configuration, no background signal, e.g.,light, can enter the detection volume 3232. Thus, when in the firstconfiguration, the detector 3212 can be calibrated and/or backgroundsignals can be detected for later use in processing the signal data.

As shown in FIG. 50, the shutter 3230 can be moved to a second shutterposition such that the channel 3209 of the housing 3202 is incommunication with the detection volume 3234. When the shutter is in thesecond shutter position, the channel 3209 of the housing 3202 is incommunication with the detection volume 3234. More particularly, in someembodiments, the shutter 3230 includes an activation surface configuredto be engaged by the second portion 12735 of the container 12700 suchthat shutter 3230 can be moved from the first position to the secondposition (FIG. 50) when the container 12700 is moved within the channel3209. In this manner, when the second portion 12735 of the container12700 is placed into communication with the detection volume 3234, theshutter 3230 is moved. Said another way, in some embodiments, theshutter 3230 can be in the first shutter position when the second endportion 12735 of the container 12700 is within the channel 3209 outsideof the detection volume 3234, and the shutter 3230 can be in the secondshutter position, when the second end portion 12735 of the container12700 is disposed within the detection volume 3234.

As shown in FIG. 49, the container 12700 can be moved from the firstposition downward (or distally) into the channel 3209 such that thesecond end portion 12735 of the container 12700 contacts the shutter3230 to urge the shutter 3230 from the first shutter position to thesecond shutter position, as shown by the arrow GG. In some embodiments,the shutter 3230 can include a ramp configured to engage the secondportion 12735 of the container 12700 when the second portion 12735 ofthe container 12700 moves within the channel 3209 towards detectionvolume 3234 to move the shutter 3230 towards the second shutterposition.

In some embodiments, the shutter 3230 can be configured to translatewithin the housing 3202 in a direction offset a longitudinal axis of thechannel 3209. In some embodiments, the detector assembly 2200 can alsoinclude a biasing member configured to urge the shutter 3230 towards thefirst shutter position.

In some embodiments, the shutter 3230 can define a calibration port (notshown). When the shutter 3230 is in the first position, the calibrationport can be aligned and/or configured place a calibration light sourcein communication with the detection volume 3234 e.g., to calibrate thedetector 3212. When the shutter 3230 is in the second position, thecalibration port can be isolated from the detection volume 3234, therebypreventing any signal leakage from the detection volume and/or ambientlight from entering the detection volume.

In some embodiments, a detector assembly, e.g., the detector assembly3200 or any other detector assembly described herein, can include ahousing defining a channel configured to receive a sample container. Thehousing can also define a seal surface and detection volume configuredto place the channel in communication with a detector. The detectorassembly can further include a shutter, e.g., shutter 3230 or any othershutter described herein, having a portion movably disposed within thehousing between a first shutter position and a second shutter position.The shutter can include a seal surface and an actuation portion which isconfigured to engage a distal end portion of the sample container tomove the shutter from the first shutter position to the second shutterposition when the distal end portion of the container is moved towardsthe detection volume. The seal surface of the shutter and the sealsurface of the housing can be configured to isolate the detection volumefrom the channel of the housing when the shutter is in the first shutterposition, while the channel of the housing can be in communication withthe detection volume when the shutter is in the second shutter position.

In some embodiments, a detector assembly, e.g., the detector assembly3200 or any other detector assembly described herein, can include ahousing defining a channel configured to receive a sample container. Thehousing also defines a detection volume configured to place the channelin communication with a detector, and further includes a seal surface.The detector assembly can also include a shutter, e.g., shutter 3230 orany other shutter described herein, shutter defining a calibration portconfigured to receive a calibration light source. The shutter can bemovably disposed within the housing between a first shutter position anda second shutter position such that a seal surface of the shutter and aseal surface of the housing is configured to isolate the detectionvolume from the channel of the housing when shutter is in the firstshutter position. Furthermore, in the first shutter position, thecalibration port can be in communication with the detection volume. Theshutter can be configured such that the channel of the housing can be incommunication with the detection volume when the shutter is in thesecond shutter position and the calibration port can be isolated fromthe detection volume.

Referring now to FIGS. 51-95, an instrument 11000 can include a housing11100, a heater assembly 11400, a drive assembly 11500, a manipulatorassembly, 11600, and a detector assembly 11200. The housing 11100, isconfigured to house, contain, and/or provide mounting for each of thecomponents and/or assemblies of the instrument 11000, as describedherein. The heater assembly 11400 is configured to receive a container,e.g., container assembly 3700, as described herein, and heat a samplecontained therein. Similarly stated the heater assembly 11400 isconfigured to maintain a sample containing target cell, at apredetermined temperature and for a desired time period (e.g., at orabove room temperature, 25 degrees Celsius, or 37 degrees Celsius, forapproximately 2 hours or 4 hours, as described herein). The driveassembly 11500 is configured to drive, transfer and/or move themanipulator assembly 11600 (and the container assembly 3700 coupledthereto) in a 3-dimensional space within the housing 11100. Said anotherway, the drive assembly 11500 can move the manipulator assembly 11600 inan X, Y and/or Z direction within the housing 11100. The manipulatorassembly 11600 is configured to manipulate, grip and/or actuate acontainer (e.g., the container assembly 3700) within the housing 11100.For example, the manipulator assembly 11600 is configured to releasablycouple to, engage, hold, lock, and/or secure the container assembly 3700to transfer the container from a first location within the housing 11100to a second location. Similarly stated, the manipulator assembly 11600and the manipulator assembly 11600 are collectively configured totransfer the container assembly 3700 between a first subassembly andanother subassembly, and/or actuate an actuation mechanism of thecontainer assembly 3700, to transfer reagents and/or solutions from oneportion of the container assembly 3700 to another. The detector assembly11200 is configured to detect a signal, e.g., luminescence, from withinat least a portion of the container assembly 3700. The detected signalcan be produced by a chemical reaction occurring within a reactionchamber of the container, for example, interaction of a reportermolecule (e.g., luciferase), with a substrate (e.g., tridecanal). Eachof these assemblies is described further below in detail, followed by adescription of various methods that can be performed by the instrument11000. Although the instrument 11000 is shown and described asmanipulating and/or actuating the container assembly 3700, theinstrument 11000 can receive, manipulate and/or actuate any of thecontainer assemblies described herein.

As shown in FIGS. 51-54, the housing 11100 is defines an internal volumeto house, contain and/or mount the subassemblies of the instrument11000. The housing 11100 can be formed from any suitable rigid, lightweight and sturdy material. Example materials includepolytetrafluoroethylene, high density polyethylene, polycarbonate, otherplastics, acrylic, sheet metal such as aluminum, any other suitablematerial or a combination thereof. The housing can be relatively smoothand free of sharp edges. The housing includes sidewalls 11102, a lid11104 and a front panel 11106. The lid 11104 is pivotally mounted on thesidewalls 11102, and can swivel about its pivot mounts from a firstposition wherein the housing 11100 is closed, to a second positionwherein the housing 11100 is open, e.g., to allow access of thesubassemblies disposed within the housing 11100. The front panel 11106defines a first opening 11108 and a second opening 11110. The firstopening 11108 and the second opening 11110 are configured to removablyreceive a series of cartridges 11300 (e.g., 1, 2, 3, 4, or even more;see FIG. 52) as described herein. Each cartridge 11300 can containand/or hold a series of container assemblies, e.g., container assembly3700. The first opening 11108 can be configured to be a loading zone,i.e. configured to removably receive the series of cartridges 11300having a series of container assemblies 3700 disposed therein. Theseries of containers can include samples for analysis in accordance withany of the methods shown and described herein. In particular, thecontainers can include samples for which screening for the presence of atarget cell (e.g., MRSA). The second opening 11110 is configured to bean unloading zone, i.e., configured to removably receive a series ofcartridges 11300 having containers disposed therein, the containershousing samples that have been analyzed by the instrument, e.g.,analyzed for the presence of bacteria using any of the subassemblies ofthe instrument 11000, and method described herein, e.g., method 400.

The housing 11100 includes an interface 11112 located on a backside ofthe housing 11100. The interface 11112 includes an electric plug 11122,e.g., for communicating electrical power to the instrument 11000. Theinterface 11112 also includes a communication interface 11124, forexample, to enable communication with an external device, e.g., localcomputer, remote computer, and/or a laboratory information system 1900,via local area network (LAN), wide area network (WAN) and/or theInternet. The communication interface 11124 can be a hardwiredinterface, e.g., DSL and/or RJ45. The housing 11110 also includes vents11114 on the backwall that are configured to allow air, e.g., air heateddue to the heat generated by operation of the subassemblies ofinstrument 11000, to exhaust. In some embodiments, the vents 11114 canincludes fans to produce a flow of the exhaust air from the instrument11000 with increased velocity, for example, to enable rapid cooling ofthe instrument. The housing 11100 also includes a series of bumpers(e.g., four, five or six) on a bottom surface configured to providecushioned seating of the instrument 11000 on a surface. The bumpers canbe made from a vibration absorbent and high friction material e.g.,rubber, and can be configured to absorb any vibrations of the instrument11000, e.g., caused by a motion of the drive assembly 11500, and/orprevent the instrument 11000 from sliding on the surface upon which itis placed.

FIGS. 55-57 show perspective views of the instrument 11000 from variousangles with the housing 11100 removed, to more clearly show the internalcomponents and subassemblies. The instrument 11000 includes a baseplate11118 configured to provide mounts for coupling the components andsubassemblies of the instrument 11000. Referring also now to FIG. 58,the instrument includes a power supply 11120 mounted on the baseplate11118. The power supply 11120 can be any commercially available powersupply as is commonly known in the arts. The power supply 11120 isconfigured to receive electric power from the electrical plug 11122,e.g., 110V at 60 Hz or 220V at 50 Hz, and convert it into an electricpower usable by the subassemblies of the instrument, e.g., step down thevoltage, step up the voltage, control current, etc.

Referring also now to FIG. 59, the instrument 11000 includes a processor11126 coupled to a frame 11127 e.g., via zip ties, and disposed on thebase plate 11118 via the frame 11127. The processor 11126 can beconfigured to control the operation of the various subassembliesincluded in the instrument 11000. For example, the processor 11126 canbe a computer, a programmable logic chip (PLC), a microprocessor, anASIC chip, an ARM chip, and/or a combination thereof. In someembodiments, the processor 11126 can included algorithms or softwarethat can include instructions for operating the instrument 11000subassemblies, e.g., heater assembly 11400, drive assembly 11500,manipulator assembly 11600, detector assembly 11200, and/or any othercomponent included in the instrument 11000. In some embodiments, theprocessor 11126 can also be programmable, e.g., configured to acceptinstructions from a user, such as operating parameters of the instrument11000. In some embodiments, the processor 11126 can also include amemory, e.g., to store the status and/or any other information (e.g.,containers analyzed, positive samples, negative samples) orinstructions.

Referring also now to FIG. 60, the instrument 11000 also includes acommunications module 11128 coupled to the mount 11129, e.g. via zipties and disposed on the base plate 11118. The communications module11128 is configured to communicate information to an external device,e.g., a laboratory information system (LIS), a remote computer,smartphone app and/or remote server from the processor 11126. In someembodiments, the communications module 11128 can also be configured toreceive instructions to facilitate the performance of the methodsdescribed herein. The communications module 11128 can employ and/or becompatible with standard communication protocols, e.g., USB, firewire,ZigBee, Bluetooth®, low powered Bluetooth®, and/or any othercommunication equipment.

As shown in FIGS. 61-63 the instrument 11000 includes a series ofcartridge receivers 11350 mounted on the baseplate 11118. Each of thecartridge receivers 11350 is configured to removably receive a cartridge11300. FIGS. 55-57 show the series of cartridges 11300 in a firstconfiguration, such that the series of cartridges 11300 are coupled tothe series of cartridge receivers 11350, and are disposed substantiallyinside the housing 11100 of the instrument 11000. FIG. 61 shows theseries of cartridges 11300 in a second configuration, wherein the seriesof cartridges 11300 are decoupled from the corresponding cartridgereceiver 11350, and are disposed substantially outside the housing 11100of the instrument 11000. Although the series of cartridges 11300 caninclude both “loading” and “unloading” cartridges 11300, as shown, thecartridges 11300 are substantially similar to each other and can be usedinterchangeably. In other embodiments, an instrument can includedifferent cartridges for loading (e.g., input) of the containerassemblies and unloading (e.g., output) of the container assemblies.Each cartridge 11300 includes a proximal end 11302 and a distal end11301. The distal end 11301 is configured to engage with and reversiblycouple to the cartridge receiver 11350. The proximal end 11302 isconfigured to allow a user to manipulate the cartridge11300, forexample, load or unload the cartridge 11300, as described herein.

FIG. 62 shows a perspective view of a cartridge 11300. The cartridge11300 can be formed from a light weight and rigid material, e.g.,plastics, and can have a surface that is smooth and relatively free ofsharp edges. The cartridge 11300 defines a series of receptacles 11304configured to removably receive at least a portion of a container, e.g.,reaction chamber 3732 of the container assembly 3700, or any othercontainer described herein. As shown herein, the cartridge 11300includes twelve receptacles 11304. In other embodiments, however, thecartridge 11300 can include 1, 2, 4, 6, 8, 10, 14, 16 or an even highernumber of receptacles 11304. Each of the series of receptacles 11304 canbe shaped and sized to receive a reaction chamber of a container, e.g.,reaction chamber 3732 of the container assembly 3700 or any othercontainer described herein, with close tolerance. In this manner, thecartridge 11300 and the receptacles 11304 can restrict lateral movementof the container. Further, receptacle 11304 of the series of receptaclescan have a depth such a reagent module, e.g., reagent module 3740 of thecontainer assembly 3700 or any other container described herein, isdisposed substantially outside the receptacle 11304. In this manner atleast a portion of the container assembly can be exposed and/oraccessible to be manipulated by the manipulator assembly 11600.

Each of the series of receptacles 11304 also includes a slot 11306 alongat least a portion of the sidewall of the receptacle 11306. In someembodiment, the slot 11306 can be configured to allow a user to access asidewall of a reaction chamber, e.g., reaction chamber 3732, of acontainer disposed within the receptacle 11304, e.g., to facilitateremoval of the container from the receptacle 11304. In otherembodiments, the slot 11306 can be configured to allow for opticalmonitoring and/or identification (e.g., via a label) of the containerassembly within the receptacle 11304.

The proximal end 11302 of the cartridge 11300 includes an arm 11308configured to be engaged by a user, e.g., to facilitateloading/unloading of the cartridge 11300 from the instrument 11000. Thearm 11308 can have an ergonomic shape, e.g., to minimize physical stresson a user manipulating the cartridge 11300.

The cartridge 11300 includes a recess 11310 that runs from the proximalend 11302 to the distal end 11301 along the entire length of thecartridge. The recess can be shaped and sized to be slidably received bya guide rail 11352 of the cartridge receiver 11350, as described herein.The distal end 11301 of the cartridge 11300 also includes a tab 11312.The tab 11312 is configured to protrude from proximal end 11312 andinterface with a sensor 11362 of the cartridge receiver 11350, asdescribed herein.

FIG. 63 shows a perspective view of a cartridge receiver 11350 includedin the instrument 11000. The cartridge receiver 11350 can be securelymounted on the base plate 11118, e.g., via screws, bolts, rivets, or thelikes. The cartridge receiver can be formed from a lightweight, rigidand wear resistant material, e.g., metals such as aluminum. Thecartridge receiver 11350 includes a guide rail 11352 configured to slideinto the recess 11310 of the cartridge 11300, such that the cartridge11300 can be slideably received by and/or coupled to the cartridgereceiver 11350. The cartridge receiver 11350 includes a latch 11354 thatcan be at least partially disposed in an opening in the guard rail11352. The latch 11354 includes a first portion 11355 configured toengage a notch 11314 (FIG. 64) included in the recess 11310 of thecartridge 11300 to lock, hold and/or secure the cartridge 11300 to thecartridge receiver 11350. The latch 11354 includes a second portion11356 that is mounted on a shaft 11361 of an actuator 11360 included inthe cartridge receiver 11350. A spring 11358 is also mounted on theshaft 11361. The spring 11358 is coupled to the latch 11354 and isoperable to urge the latch 11354 to lock and/or engage the cartridge11300 as described herein. The spring 11358 can be, e.g., a tensionspring, such as e.g., a helical tension spring. The cartridge receiver11350 includes the sensor 11362. The sensor 11362 can be any suitablesensor, such as a motion sensor, a position sensor, an optical sensor, apiezoelectric sensor, or any other suitable sensor. As described above,the sensor 11362 is configured to interface with the tab 11312 of thecartridge 11300 to determine and/or validate a position of the cartridge11300, e.g., to ensure that the cartridge 11300 is completely in thesecond configuration, and is fully coupled to the cartridge receiver11350.

FIG. 64 shows a side cross-section of a cartridge 11300 in the secondconfiguration, such that the cartridge 11300 is fully coupled with thecartridge receiver 11350. In the second configuration, the guide rail11352 of the cartridge receiver 11350 is disposed substantially into therecess 11310, and the first portion 11355 of the latch 11354 is insertedinto the notch 11314 included in the recess 11310 of the cartridge11300. The latch 11354 is pivotally mounted on a pin 11364, such thatthe latch 11354 can pivot about the pin 11364 from a first position to asecond position. In the first position, at least a portion of the firstportion 11355 of the latch 11354 is inside the notch 11314. In thesecond position the first portion 11355 is outside the notch 11355. Thespring 11358 is coupled to the second portion 11356 of the latch 11354,and is operable to urge the latch 11354 into first position.

In use, the cartridge 11300 can be loaded into the instrument 11000 bysliding the cartridge along the guide rail 11352 until the proximal end11301 of the cartridge 11300 contacts the first portion 11355 of thelatch 11354. The first portion 11355 of the latch 11354 has a taperedsurface, such that an edge (e.g., a chamfered or tapered edge) of theproximal portion 11301 of the cartridge 11300 slides along the taperedsurface of the first portion 11355 of the latch 11354, urging the latch11354 to pivot from the first position to the second position. As thecartridge 11300 moves further along the guide rail 11352 towards thesecond configuration, the first portion 11355 of the latch 11354encounters the notch 11314. The spring 11358 now urges the latch 11354to pivot about the pin 11364 and move back into the first position suchthat the first portion 11355 of the latch 11354 is inside the notch11314 and cartridge 11300 is locked in the second configuration.

In the second configuration, the tab 11312 engages the sensor 11362, asdescribed above. In some embodiments, the sensor 11362 produce a signalindicating that the cartridge 11300 is fully coupled to the cartridgereceiver 11350. The sensor 11362 can communicate the informationvalidating the position of the cartridge 11300 to the processor 11126and/or the user, e.g., using audio (e.g. beeps), visual (e.g., indicatorlights) and/or tactile alerts. The actuator 11360 is configured to urgethe latch 11354 from the first position to the second position torelease the cartridge 11300 for removal from the instrument. Forexample, a user can actuate the actuator 11360 and/or the actuator 11360can be configured to actuate after a given time period, such that theshaft 11361 extends towards the proximal end 11302 of the cartridge11300. This causes the latch 11354 to pivot about the pins 11364 fromthe first position to the second position, so that the cartridge 11300is no longer secured by the latch 11354 and can be slideably removedfrom the cartridge receiver 11350.

As shown in FIG. 55-57, the instrument 11000 includes a heater assembly11400, configured to receive a series of containers, e.g., containerassembly 3700 and/or any other container described herein. The heaterassembly 11400 is configured to heat and/or maintain a temperature ofthe container assemblies therein according to any of the methodsdescribed herein. Referring now also to FIG. 65-67, the heater assembly11400 includes a housing 11410 configured to house a series of heatingblocks 11420. The housing 11410 is formed from a rigid, and heatinsulating material such as thick stainless steel, other metals,polymers. The housing 11410 can include a lining of a heat resistantmaterial, e.g., Mica, or any other heat insulation means. The housing11410 defines a series of cavities 11412, each being sized and shaped toreceive a heating block 11420 with close tolerance.

The heating block 11420 can be formed from a heat conducting, rigid andwear resistant material, e.g., anodized aluminum. The heating block11420 defines a series of receptacles 11422, each of which is shaped andsized to receive at least a portion of a container, e.g., reactionchamber 3732 of the container assembly 3700, or any other containerdescribed herein. For example, a container can be disposed on any of theseries of receptacles 11422 of any of the series of heating blocks 11420by the manipulator assembly 11600, which is driven and/or positioned bythe drive assembly 11500 as described herein.

Each heating block 11420 includes heating elements 11424 disposed on abottom surface of the heating block 11420. In some embodiments, theheating element 11424 can be a micro heater, e.g., a ceramic plateheater. In some embodiments, the heating elements 11424 can beelectrical wires that pass a current through the heating block 11420 toheat the heating block 11420 using electrical energy (i.e., resistiveheating). Each heating block 11420 also includes a temperature sensor11426, e.g., a thermocouple, disposed within a body of the heating block11420. The temperature sensor 11426 is in electrical communication withthe heating element 11420 directly, or through a processing unit (notshown) of the instrument 11000. In this manner, the heating elements11424 can be deactivated and/or controlled to limit heating to theheating block 11420, e.g., when the temperature of the sensors exceeds apredefined temperature level. In this manner, the temperature of theheating block 11424, and the samples disposed therein can be controlledin accordance with the methods disclosed herein.

As shown in FIG. 55-57, the instrument 11000 includes a drive assembly11500 configured to transport a container or assembly coupled thereto,e.g., by the manipulator assembly 11600, as described herein. Forexample, the manipulator assembly 11600 can be coupled to a container,and the drive assembly 11500 can be configured to enable transport ofthe container from a first location within the housing 11100 to a secondlocation, e.g., from a loading cartridge 11300 to the heater assembly11400, from the heater assembly 11400 to the detector 11200, from thedetector 11200 to an unloading cartridge 11300, and/or any otherlocation therewithin.

Referring also now to FIG. 68-71, the drive assembly 11500 includes asupport 11502, e.g., a frame or a chassis, on which the components ofthe drive assembly 11500 are mounted. The support 11502 is securelymounted on the baseplate 11118, and can be configured to absorb anyvibrations caused by the motion of the drive assembly 11500. The supportincludes a first section 11503 a that is oriented along an X axis withrespect to the X axis of the instrument 11000, as shown by the arrow XX,and a second section 11503 b oriented along a Y-axis with respect to theinstrument 11000, as shown by the arrow YY. The second section 11503 bis movably disposed on and/or coupled to the first section 11503 a viathe second guide block 11520. The drive assembly 11500 includes a firstactuator 11504 a disposed on the first section 11503 a, a secondactuator 11504 b disposed on the second section 11503 b, and a thirdactuator 11504 c mounted on the frame 11512. The first actuator 11504 aand the second actuator 11504 b are configured to drive the frame 11512and any subassembly, e.g., the manipulator assembly 11600, disposed onthe mount 11514, in an X and Y direction, respectively, as describedherein. The third actuator 11504 c is configured to drive the mount11514 and any subassembly mounted thereon, e.g., the manipulatorassembly 11600, in a Z direction with respect to the instrument 11000,as shown by the arrow ZZ. The actuators can be substantially similar toeach other and can include, e.g., stepper motors, configured to drivethe frame 11512 and/or mount 11514 a fixed distance with every step,e.g., each portion of a rotation of the actuators 11504 a, 11504 band/or 11504 c.

The first actuator 11504 a and the second actuator 11504 b include afirst disc 11506 mounted on each of the actuators 11504 a, 11504 b. Asecond disc 11508 (FIG. 70) is disposed on each of the first section11503 a and the second section 11503 b of the support 11502. A belt11510 is looped around the first disc 11506 and the second disc 11508 ina taut manner, such that a rotation of the disc 11506 caused by theactuator 11504 a/b urges the belt 11510 to be driven along its length(or rotated) over the second disc 11508. The belt 11510 can be, e.g., arubber belt, a plastic belt or a polymer belt, and can include grooveson the surface contacting the discs 11506 and 11508, e.g., to providefriction and/or no slip translation of the belt. The belt 11510 coupledto the first actuator 11504 a is operably coupled to a first guide block11516 which is mounted on a first guide rail 11518. The belt 11510 isconfigured such that translation of the belt 11510 caused by the firstactuator 11504 a urges the first guide block 11516 and the secondsection 11503 b of the support 11502 mounted thereon, to translateslidably along the guide rail 11518 in the X direction. Similarly, thesecond actuator 11504 b also includes a belt 11510 disposed on the disc11506 which is coupled to the second disc 11508 disposed on the frame11512. The frame 11512 is coupled to the second guide block 11520. Thesecond guide block 11520 is mounted on a second guide rail 11522.Driving the belt 11510 by the second actuator 11504 b, drives the frame11512 and the second guide block 11520 slidably along the second guiderail 11522. In this manner, a combination of translation of the secondsection 11503 of the support 11502 caused by the actuation of the firstactuator 11504 a along the X axis, and the translation of the frame11512 along the Y axis caused by the actuator 11504 b, can drive theframe to any location in an X-Y plane within the instrument 11000.

The frame 11512 is coupled to a first chain 11524 a slidably disposed ina first chain guide 11526 a, which is disposed on and coupled to thefirst section 11503 a. A second chain 11524 b is also coupled to theframe 11512, and is slidably disposed in a second chain guide 11526 b,which is disposed on and coupled to the second section 11503 b. Thechains 11524 a/b are configured to slide along the chain guides 11526a/b in close tolerance corresponding to an X-Y displacement of the frame11512, such that the chains prevent any lateral motion of the frame11512, e.g., to ensure accurate displacement of the frame 11512.

As described herein, the third actuator 11504 c is disposed on the frame11512 and includes a first disc 11528 a coupled to the actuator 11504 c.A belt 11530 is coupled to the first disc 11528, such that the belt11530 loops over the first disc 11528 a and a second disc 11528 b. Thesecond disc 11528 b is coupled to a lead screw 11532 included in theframe 11512. The belt 11530 can be substantially similar to the belt11510. The lead screw 11532 has a nut 11534 mounted on threads of thelead screw 11532. The nut is coupled to the mount 11514. The thirdactuator 11504 c is configured to rotate the first disc 11528 a whichurges the belt 11530 to translate, thus rotating the second disc 11528b. Rotation of the second disc 11528 b rotates the lead screw 11532 thaturges the nut 11534 to displace along the length of the lead screw 11532from a first position (FIG. 70), to a second position (FIG. 71) in the Zdirection. Displacement of the nut 11534 also causes the mount 11514coupled to the nut 11534, and any subassembly mounted thereon, e.g., themanipulator assembly 11600, to move from the first position to thesecond position in the Z direction. The mount 11514 is also coupled to athird guide block 11536 slidably mounted on a third guide rail 11538,such that displacement of the mount 11514 in the Z direction is guidedby the third guide block 11536 and the third guide rail 11538, e.g., toprevent any radial motion of the mount 11514 about a longitudinal axisdefined by the lead screw 11512.

The drive assembly 11500 includes sensors 11540 mounted each of thefirst section 11503 a, the second section 11503 b, and the frame 11512.The sensors 11540 can be position sensors such as, e.g., opticalsensors, motion sensors, piezoelectric sensors, or any suitable sensor.The sensors 11540 can be configured to detect a location of the secondsection 11503 b and the frame 11512, e.g., to prevent an overtravel thatcan damage the drive assembly and/or cause increased wear. As shown inFIG. 57 and FIG. 72, the instrument 11000, also includes circuitry 11500for controlling the actuators 11504 a/b/c. The circuitry 11500 can beany commercially available circuitry used for control of actuators 11504a/b/c, e.g., stepper motor controller circuitry.

As shown in FIG. 55-57, the instrument includes a manipulator assembly11600 disposed on the mount 11514 included in the drive assembly 11500.The manipulator assembly 11600 is configured to grasp, hold, clamp,contact, engage and/or otherwise secure a container, e.g., containerassembly 3700 as described herein. For example, the manipulator assembly11600 can be configured to releasably engage and/or grip containerand/or engage one or more actuators included in the container, such asfor example the container assembly 3700.

Referring now to FIGS. 73-84, the manipulator assembly 11600 includes anarticulation subassembly 11610, a plunger subassembly 11630 and anactuator subassembly 11650. The articulation assembly 11610 isconfigured to releasably contact, grasp or otherwise secure a container,e.g., container assembly 3700. For example, the articulation assembly11610 can be configured to secure or grasp a container disposed in afirst location, such as the loading cartridge 11300. The articulationsubassembly 11601 can continuously secure the container during transportfrom the first location to a second location within the instrument 11000by the drive assembly 11500. Such changes in position can include movingthe container assembly from the loading cartridge 11300 to the heaterassembly 11400, from the heater assembly 11400 to the detector assembly11200 and/or from the detector assembly 11200 to the unloading cartridge11300. The plunger subassembly 11630 is configured to contact, engage orotherwise manipulate one or more actuators in a container, e.g.,actuator 3750 and/or 3760 included in the container assembly 3700, tourge the actuator and/or actuators to communicate a reagent (e.g.,biologic or abiologic vectors such as transduction particles of the typeshown and described herein) and/or a substrate (e.g., tridecanal) from areagent volume to a reaction chamber of the container, as describedherein. The actuator subassembly 11650 is configured to engage ormanipulate the plunger subassembly 11630, e.g., to manipulate an innerplunger 11632 and/or an outer plunger 11636 of the plunger assembly11630, as described herein. The actuator subassembly 11650 can also beconfigured to engage or manipulate the articulation subassembly 11610,e.g., to manipulate or otherwise urge the articulation subassembly 11610to secure or release a container, e.g., container assembly 3700, asdescribed herein. Similarly stated, the actuator subassembly 11650 can,with a single actuator, cause the manipulator assembly 11600 to bothgrip and/or secure a container, and actuate the container.

As shown in FIGS. 74-75, the articulation subassembly 11600 includes aset of grippers 11612, coupled or mounted on a set of gripper bases11614. As shown two grippers 11612 are coupled to a single gripper base11614. In some embodiments, each gripper base 11614 can include morethan two grippers 11612, e.g., three or four. The grippers 11612 areconfigured to be elongated, pin-like members that can be formed from astrong and rigid material, e.g., metals such as stainless steel. In someembodiments, the grippers 11612 can include a surface that can have ahigh friction for contacting, grasping or otherwise securing acontainer, e.g., container assembly 3700. For example, the surface ofthe grippers 11612 can include grooves, abrasions, rubber coating, softplastic and/or rubberized carbon. Each gripper base 11614 defines agroove (or channel) 11615 configured to allow a plunger portion 11637 ofthe outer plunger 11636 included in the plunger subassembly 11630, tomove within a region between the grooves 11615, without contacting asidewall defining the grooves 11615.

Each gripper base 11614 is coupled to a side plate 11616 by any suitablemechanism. The side plates 11616 are pivotally coupled to an actuatormount 11654 included in the actuator assembly 11650, as describedherein. The side plates are configured to pivot or articulate abouttheir mounts relative to the plunger assembly 11630, to urge thearticulation assembly 11610 from a first configuration to a secondconfiguration. In the first (or closed) configuration the set ofgrippers 11612 are in a closed position to selectively engage, grip,grasp or otherwise secure a container, e.g., container assembly 3700. Inthe second (or opened) configuration the set of grippers 11612 aredistal to each other, e.g., to disengage or release the container. A setof guide wheels 11620 is disposed on a side wall of the each of the setof side plates 11616, e.g., mounted on pins, bolts, screws or the likes.Under certain conditions, the set of guide wheels 11620 is configured tobe proximate to but not contacting, or in contact with a portion of aside wall of a set of guide members 11640 included in the plungerassembly 11630, as described herein. The guide members 11640 areconfigured to urge the side plates 11616 and thus the articulationassembly 11610 from the first configuration to the second configuration,as described herein.

The side plates 11616 are coupled to a set of compression plates 11618.The compression plates 11618 are in pressure contact with a set ofsprings 11622, which are mounted to the actuator mount 11654 via pins.The set of compression plates 11618 are configured to angularly displacewith the pivotal motion of the set of side plates 11616, such that theset of compression plates 11618 engage the set of springs 11622, e.g.,compress the springs 11622, when the articulation assembly 11610 is inthe second configuration. Thus the compression plates 11618 areconfigured to urge the articulation assembly 11610 into the firstconfiguration from the second configuration, e.g., to grasp or secure acontainer, e.g., container assembly 3700 or any other containerdescribed herein. The springs 11622 are configured to control the amountof force exerted by the grippers 11612 on the container, e.g., toprevent crushing of the container. At least one of the set of sideplates 11616 can be configured to engage a position sensor 11662 a,e.g., an optical sensor, motion sensor, piezoelectric sensor, or anyother suitable sensor. The sensor 11662 a is coupled to a sensor mount11664, which is mounted a surface of the actuator mount 11654. Thesensor 11662 a can be configured to inform a control system and/or userabout the position of the articulation assembly 11610, e.g.,articulation assembly in first configuration (i.e., securing, engagingor grasping a container) or second configuration (i.e., disengaging orreleasing a container). In some embodiments, the sensor 11662 a can be ahoming sensor, configured to identify a home position of thearticulation assembly 11610.

The plunger subassembly 11630 includes an inner plunger 11632 and anouter plunger 11636. As shown, the inner plunger 11632 is also a leadscrew of the actuator 11652. Accordingly, the inner plunger 11632 canrotate about a longitudinal axis of the plunger assembly 11630. Theinner plunger 11632 can be configured to engage or manipulate a firstactuator of a container, e.g., the actuator 3750 of the containerassembly 3700 as described herein. The outer surface of the innerplunger 11632 is threaded and can be threadedly disposed within a collar11634. The collar 11634 is configured to move along the threads of theinner plunger 11632 during the rotation of the inner plunger 11632. Thecollar 11634 is further coupled to an engagement portion 11637 of theouter plunger 11636, such that a rotation of the inner plunger 11632results in a longitudinal displacement of the outer plunger 11636. Thiscan, for example, allow control of the speed of the outer plunger 11636,which can be used to control delivery of a substrate into a reactionchamber of a container, as described herein. The outer plunger 11636includes an engagement portion 11638 which can be configured to engageor manipulate a second actuator in a container, e.g., actuator 3760 ofthe container assembly 3700, as described herein. The outer plunger11636 also includes a channel 11639 defined therethrough along alongitudinal axis of the outer plunger 11636. At least a portion of theinner plunger 11632 can be disposed in the channel 11639.

A set of guide members 11640 are disposed on a side wall of the outerplunger 11636. Each of the set of guide members 11640 includes a firstsection 11642 having a first width, a second section 11644 having asecond width greater than the first width, and a third section 11643which is angled with respect to a longitudinal axis of the guide member11640 and connects the first section 11642 to the second section 11644.At least a portion of the plunger assembly 11630 e.g., the collar 11634,the outer plunger 11636 and the guide members 11640, are configured tomove along a longitudinal axis defined by the inner plunger 11632.Furthermore, the plunger assembly 11630 is configured to engage and ormanipulate a container, e.g., container assembly 3700, that is engaged,secured or otherwise grasped by the articulation assembly 11610, asdescribed herein. The plunger assembly 11630 is also configured tomanipulate the articulation assembly 11610, e.g., to urge thearticulation assembly 11610 from the first configuration, in which thearticulation assembly 11610 is engaging, grasping or otherwise securinga container, to the second configuration, in which the articulationassembly disengages or releases the container, as described below. Aclip 11646 is also disposed on one of the guide members 11640 (FIG. 75).The clip 11646 is configured to selectively engage a sensor 11662 b tovalidate and/or determine a position of the plunger assembly 11630. Thesensor 11662 b can be any suitable sensor, e.g., a position sensor, amotion sensor, an optical sensor, a piezoelectric sensor or any othersuitable sensor, disposed on the actuator mount 11654. The sensor 11662b can be used to determine a position of the plunger assembly 11630,e.g., to prevent overtravel of the plunger assembly 11630.

The actuator subassembly 11650 includes an actuator 11652, e.g., astepper motor, which is disposed on the actuator mount 11654. Theactuator mount 11652 defines corresponding recesses 11656 a and 11656 b.The recess 11656 a provides a mounting seat for at least a portion ofthe actuator 11652. The groove 11656 b defines an opening through whichat least a portion of the plunger assembly 11630, e.g., the innerplunger 11632, the collar 11634 and the outer plunger 11636 can move.The actuator mount 11654 further includes a set of alignment pins 11658disposed on a bottom surface of the actuator mount 11654. The alignmentpins 11658 are substantially parallel to the longitudinal axis definedby the plunger assembly 11630. At least a portion of each alignment pin11658 is disposed in a corresponding channel 11659 included in thecollar 11634 and the engagement portion 11637 of the outer plunger11636. In this manner, the displacement of the plunger subassembly 11630along the longitudinal axis defined by the plunger subassembly 11630,e.g., caused by a rotation of the inner plunger 11632, is guided by thealignment pins 11658, e.g., to prevent any rotation or sideways motionof the plunger assembly 11630.

As described herein, the manipulator assembly 11600 is configured toreleasably contact, engage or otherwise secure a container. FIG. 75shows a side view of the manipulator assembly 11600 in a firstconfiguration, in which the housing 3741 of the container assembly 3700is not contacted, grasped or otherwise secured by the manipulatorassembly 11600. FIG. 75 shows a side view of the manipulator assembly11600 in a second configuration, in which the manipulator assembly 11600is securing or gripping the container assembly 3700. The manipulatorassembly 11600 is configured such that the grippers 11612 secure thecontainer assembly 3700 only from the top portion, i.e., the reagentmodule 3740. This can allow a bottom read of the container assembly 3700by the detector 11200 or any other detector described herein.

As shown in FIG. 75, when in the first configuration, the actuator 11652has rotated the inner plunger 11632 to move the collar 11634 coupled tothe inner plunger 11632 along the length of the inner plunger 11632 in adirection towards the actuator 11652. In this manner, when in the firstconfiguration the actuator 11652, at least a portion of the collar 11634and/or the outer plunger 11636 coupled to the collar 11634, are withinthe opening defined by the recess 11656 b of the actuator mount 11654.Moreover, the position of the plunger assembly 11630 relative to theactuator assembly 11650 (i.e., in an upward position) is such that theguide members 11640 engage the articulation assembly 11610. As shown inthe first configuration illustrated in FIG. 75, the displacement of theguide member 11640 towards the actuator 11652 places the guide wheels11620 in contact with the second section 11644. In this configuration,the set of side plates 11616 pivot or articulate about their pivotmounts relative to the longitudinal axis of the plunger assembly 11630,such that the gripper bases 11614 and the set of grippers 11612 areangled with respect to the longitudinal axis of the plunger assembly11630. An end of corresponding grippers 11612 is a first distance apart,such that the first distance is greater than a diameter of the housing3741 of the container assembly 3700.

In the first (or “open grip”) configuration, the manipulator assembly11600 is configured to disengage or release the container assembly 3700and/or is ready to receive the container assembly 3700. For example, insome embodiments, the container assembly 3700 can be disposed in acartridge 11300. The manipulator assembly 11600 can be driven by thedrive assembly 11500 to the location where the container assembly 3700is disposed, and then urged into the first configuration. Themanipulator assembly 11600 can then be displaced along a longitudinalaxis defined by the manipulator assembly 11600 towards the containerassembly 3700, until the grippers 11612 are adjacent to the housing3741, and a bottom portion of the inner plunger 11632 is in closeproximity to but not contacting an engagement portion 6752 of an innerplunger 6750 (not shown in FIG. 75-76) disposed in the housing 3741 ofthe container assembly 3700.

As shown in FIG. 76, the articulation assembly 11610 can be moved intothe second configuration to engage, grip, grasp or otherwise secure thecontainer assembly 3700. For example, to move the articulation assemblyto the second configuration, the inner plunger 11632 is rotated by theactuator 11652. This urges the collar 11634 to move along the length ofthe inner plunger 11632 in a direction away from the actuator 11652(e.g., downward as shown in FIG. 76). Displacement of the collar 11634also urges the outer plunger 11636 and the set of guide members 11640coupled thereto, to move along a longitudinal axis defined by theplunger assembly 11630 away from the actuator 11650. This causes theguide wheels 11620 to ride along a side wall of the second section 11644of the guide member 11640 onto the narrower inclined section 11643. Inthis configuration, the set of springs 11622 that are in pressurecontact with the set of compression plates 11618, urge the set ofcompression plates and hence the set of side plates 11616 to pivotrelative to the longitudinal axis defined by the plunger assembly 11630.This also causes the gripper bases 11614 and the set of grippers 11612coupled thereto to displace towards the housing 3741 of the containerassembly 3700, such that in the second configuration, the set ofgrippers 11612 are contacting, engaging or otherwise securing thecontainer assembly 3700. The manipulator assembly 11600 can bemaintained in the second configuration to transport the containerassembly 3700 from a first location within the housing 11100 of theinstrument 11000 to a second location as shown in FIG. 77, via the driveassembly 11500.

As described herein, the manipulator assembly can also be used to engageand or manipulate the container assembly 3700, e.g., the actuators 3750and 3760 disposed in a housing 3741 of the container assembly 3700.FIGS. 78 and 79 show side views of the manipulator assembly 11600 in thefirst configuration (also referred to as “open grip”) and FIG. 80 showsa side cross section of the manipulator assembly 11600 shown in FIG. 78.In this configuration, the plunger assembly 11630, the articulationassembly 11610 and the actuator assembly 11650 are in the same relativepositions as discussed above with regard to FIG. 75. The positioning ofthe manipulator assembly 11600 relative to the container assembly 3700,however, is different. In particular, the inner plunger 11612 engagesthe engagement portion 3752 of the actuator 3750 as described belowherein.

In this configuration (the “inner plunge” configuration), the containerassembly 3700 can be disposed e.g., in a recess 11412 of a heating block11420 included in the heater assembly 11400 as described herein, suchthat the container assembly 3700 cannot be displaced laterally withrespect to a longitudinal axis of the manipulator assembly 11600. Whenthe articulation assembly 11610 is in the first configuration (“opengrip” and the “inner plunge”), at least a portion the collar 11634 andthe outer plunger 11636 is located within the opening defined by therecess 11656 b such that a bottom portion of the inner plunger 11632 isprotruding from the bottom of the channel 11639 of the outer plunger11636. Moreover, as described above, the set of guide wheels 11620 arecontacting the surface 11644 of the set of guide members 11640 and theset of compression plates 11618 are compressing the set of springs 11622to maintain the grippers in position.

To execute the “inner plunge” operation as shown in FIG. 78-80, thedrive assembly 11500 is actuated to displace the manipulator subassembly11600 in a downwards direction defined by a longitudinal axis of theplunger assembly 11630, as shown by the arrow ZZ. The downwards motionof the manipulator assembly 11600 causes the inner plunger 11632 to movedownwards, such that a bottom portion of the inner plunger 11632contacts the engagement portion 3752 of the actuator 3750, urging theplunger portion 3754 of the actuator 3750 within the internal volume3742 defined by the housing 3741. The plunger portion 3754 can move froma first position to a second position, such that the plunger portion3754 communicates a reagent disposed in the internal volume 3742 intothe container assembly 3700, as described above. The reagent can be anyreagent or substance described herein, such as a biologic or abiologicvector or transduction particle of the types described herein. Afteractuation of the first actuator 3750 by the manipulator assembly 11600,the container assembly 3700 can remain disposed in the heater assembly11400 for a predefined time period and at a predetermined temperature.As described above, in some embodiments, maintaining the containerassembly will allow a target cell within the sample to express asufficient quantity of reporter molecules such as, for example,luciferase or any other reporter molecule described herein.

FIG. 81 shows a side cross-section of the manipulator assembly 11600 inthe second configuration (also referred to as “gripper closed”). In thesecond configuration, the manipulator assembly 11600 is in contact with,engaged, grasped or otherwise secured to the container assembly 3700, asdescribed herein with reference to FIGS. 76 and 77. In the gripperclosed configuration, the inner plunger 11632 is disposed substantiallywithin the outer plunger 11636, such that the bottom portion of theinner plunger 11632 is proximal to but not contacting the engagementportion 3752 of the first actuator 3750 disposed in the housing 3741 ofthe container assembly 3700, as described herein. Furthermore, the setof guide wheels 11620 are contacting the third section 11643 of the setof guide members 11640. The gripper closed configuration can be used totransport the container assembly 3700, e.g., from the heater assembly11400 to the detector assembly 11200. The articulation assembly 116500is also configured to prevent overtravel. For example, if the in theclosed grip configuration, the set of guide wheels contact the secondsurface 11644 of the set of guide members 11640, this indicates theouter plunger 11636 has overtravelled. In this situation, a pin (notshown) mounted on one of the set of side plates 11616 triggers theposition sensor 11662 a, that can send an overtravel signal, e.g., analarm, to the processor 11126 or a user.

Referring now to FIGS. 82-84, FIGS. 82 and 83 show a side view of themanipulator assembly 11600 and FIG. 84 shows a side cross-section ofview shown in FIG. 82, in a third configuration (also referred to as“substrate plunge”). In the substrate plunge configuration, the outerplunger engages the engagement portion 3762 of the actuator 3760 asdescribed below. In this configuration, the container assembly 3700 canbe disposed, e.g., in the detector assembly 11200 as described herein,such that the container assembly 3700 cannot be displaced laterally withrespect to a longitudinal axis of the manipulator assembly 11600. Forexample, in this configuration, the reaction chamber 3732 of thecontainer assembly 3700 can be disposed in a slot 11234 of a shutter11230 included in the detector assembly 11200, as described furtherbelow herein. Furthermore, when moved to the third configuration, thearticulation assembly 11610 can be maintained in an engage, clamp, gripor otherwise secure configuration. In this manner, the containerassembly 3700 can remain secured by the articulation assembly 11610during the substrate plunge. This can, for example, ensure that allforce of the outer plunge is transferred only to the second actuator3760 of the container assembly 3700 and/or to maintain the containerassembly 3700 flush with a gasket 11206 included in the detectorassembly 11200 to prevent any ambient noise (e.g., light) from enteringthe detector assembly 11200, as described herein.

To move from the second configuration to the third (“substrate plunge”)configuration, the inner plunger 11632 rotates in a direction oppositeto the open grip configuration, e.g., as shown by the arrow RR in FIG.82, such that the collar 11634 displaces along the longitudinal axisdefined by the inner plunger 11632 in a direction away from the actuator11652. Displacement of the plunger also displaces the outer plunger11636 and the set of guide members 11640 attached thereto, in the samedirection. Displacement of the outer plunger 11636 effected by therotation of the inner plunger 11632 is continued such that a bottomportion of the outer plunger 11636 engages the engagement portion 3762of the second actuator 3760 urging the plunger portion 3764 of theactuator 3760 to displace within the internal volume 3744 defined by thehousing 3741 of the container assembly 3700. The plunger portion 3764can move from a first position to a second position, such that theplunger portion 3764 communicates a reagent disposed in the internalvolume 3742 through the delivery portion 3770 into the reaction chamber3732 included in the container assembly 3700. For example, the reagentcan be a substrate, e.g., tridecanal or any other substrate describedherein, that can be formulated to react with reporter molecules presentin the reaction chamber 3732, e.g., luciferase or any other reportermolecule, as described above. The reaction of the substrate withreporter molecules can produce a signal, e.g., luminescence or any othersignal described herein, which can be detected by the detector assembly11200, as described further below herein. Longitudinal displacement ofthe outer plunger 11636 effected by a rotary motion of the inner plunger11632 can allow, for example, better control over the displacement ofthe outer plunger 11636, and therefore better control over communicationof a substrate from the internal volume 3744 to the reaction chamber3732 of the container assembly 3700.

It is to be noted that a single actuator 11652 is employed by themanipulator assembly 11600 to perform a series a functions, including:i) manipulating outer plunger 11636 by actuating the inner plunger 11632to cause the substrate plunge and; ii) manipulating the guide members11640 coupled to the outer plunger 11636 to urge the articulationassembly 11610 from the open grip to the gripper closed configuration. Asecond actuator, e.g., actuator 11504 c included in the drivesubassembly 11500 as described herein, can be used to displace themanipulator assembly 11600 in the Z-direction. In some embodiment, thereagent plunge functionality can be included in the actuator 11652,e.g., the actuator 11652 can also be capable of linearly displacing theinner plunger 11632.

As shown in FIG. 55-57, the instrument includes a detector assembly11200. The detector assembly 11200 is configured to detect a signal,e.g., luminescence, produced by a chemical reaction, e.g., interactionof a reporter molecule (e.g., luciferase) with a substrate (e.g.,tridecanal) in accordance with any of the methods described herein. Thedetector assembly 11200 is configured to receive a container assembly3700, and detect a signal produced therein. The detector assembly 11200can include any suitable detector that can detect the signal produced bya reporter molecule, e.g., luciferase. For example, as shown, thedetector is an optical detector. In some embodiments a detector can be afluorescence detector (e.g. to detect a fluorescent reporter moleculesuch as GFP, etc.), a luminescence detector (e.g. to detectbioluminescence produced by a reporter molecule such as luciferase),color detector (e.g. to detect a colored precipitant produced by areporter enzyme such as HRP), a spectrometer, and/or an image capturedevice. In some embodiments, the detector can further include a lightsource. Although described as being primarily based on opticaldetection, in some embodiments, the detector can be an electrochemicaldetector. For example, the detector can include an amperometricdetector, potentiometric detector, conductometric, and/or impedometricdetector, configured to detect a current, voltage, or conductance,resistance/impedance change produced by the reporter, e.g., luciferase.In some embodiments employing electrochemical detection, the detectorcan be configured to come in physical contact with the sample solutionthat can contain a sample. In some embodiments, the detector 11200 canuse other detection methods, e.g. surface acoustic wave, surface plasmonresonance, Raman spectroscopy, magnetic sensors, and/or any othersuitable detection method known in the art.

In some embodiments, the detector assembly 11200 can only provide aqualitative answer, for example, a YES/NO answer on the presence oftarget cell. In some embodiments, the detector 11200 can quantify thetarget cell, for example, determine the cfu/ml of target bacteria in thesample according to any of the methods described herein, e.g., method400. In some embodiments, the detector assembly 11200 can include an endread system, .e.g., to allow flexible placement of a label on thecontainer, e.g., container assembly 3700. In some embodiments, the endread system includes direct contact and/or proximal disposal of atransparent end of a container, e.g., container assembly 3700 with thedetector 11200, e.g., to minimize optical instruments and/or backgroundsignal interference. In some embodiments, the detector assembly 11200 isdevoid of an incident light source. Said another way, no external lightis needed for signal detection from the reporter molecules, e.g.,luciferase, produced by a target cell, e.g., bacteria, disposed in thecontainer, e.g., container assembly 3700.

Referring now also to FIGS. 85-94, the detector assembly 11200 includesa detector 11212 and a shutter 11230 disposed in a housing 11202. Thedetector assembly 11200 is configured to removably receive a container,e.g., container assembly 3700, and detect a signal produced therein,e.g., a luminescence signal resulting from a chemical interaction of areporter molecule (e.g., luciferase) with a substrate (e.g.,tridecanal).

The housing 11202 can be formed from a strong, rigid and substantiallyopaque material, e.g., metals. In some embodiments, the housing can bepainted a dark color, e.g., black, to minimize internal reflectionsand/or refractions because of a luminescence reaction produced in acontainer disposed in the detector assembly, e.g., container assembly3700. The housing 11202 is also configured to prevent external lightfrom entering the detector assembly 11200, e.g., to reduce backgroundnoise and/or signal quality. The housing 11202 includes a groove 11203.A receptacle 11204 is disposed in the groove 11203 which defines anopening 11207 sized and shaped to removably receive a container, e.g.,container assembly 3700. The receptacle 11204 can be fixedly orremovably coupled to the housing 11202, e.g. via screws, bolts, rivets,glues, hot welded, or snap fitted in to the groove 11203 of the housing11212. As shown in FIG. 86, the receptacle 11204 includes a gasket 11206fixedly disposed in the receptacle 11204. The gasket 11206 can be formedfrom a rigid and crush and/or wear resistant material, e.g., highdensity neoprene. The gasket 11206 is configured such that when thecontainer assembly 3700 is disposed in the receptacle 11204, the gasket11206 and a portion of the reagent module 3740 form a light-tight sealto prevent any external light from entering inside the housing 11202. Insome embodiments, the height of the receptacle 11204 can be varied,e.g., to accommodate containers of various lengths. This can ensure thatany variations in length of the container, e.g., because of variationsin the manufacturing process and/or user preference, does not change thedistance of a bottom end of the container, e.g., container base, fromthe detector 11212 e.g., to enhance signal quality and/or repeatability.The housing 11202 further defines a channel 11209 configured to receiveat least a portion of the container, e.g., a reaction chamber 3732 ofthe container assembly 3700. The housing 11202 also includes an internalvolume 11210 configured to house the shutter 11230, such that theshutter 11230 is free to be manipulated and/or displaced from a firstposition to a second position within the internal volume 11210. Thehousing 11202 further includes circuitry 11208 disposed on a sidewall ofthe housing 11202. The circuitry is configured to control the operationof a light source 11246, as described below herein.

FIG. 87 shows the internal components of the detector assembly 11200with the housing 11202 removed and FIG. 88 shows an exploded view of thecomponents of the detector assembly 11200. As shown herein, the detectorassembly 11200 includes a baseplate 11211 configured to mount thedetector assembly 11200 on the baseplate 11118 of the instrument 11000.The base plate 11211 is also configured to provide a base for mountingthe components of the detector assembly 11200 and the housing 11202.

The detector assembly 11200 includes a detector 11212 disposed in adetector enclosure 11214. The detector 11212 can be an optical detector,e.g., a photomultiplier tube (PMT), a luminometer, a spectrophotometer,fluorescence detector, and or any other suitable optical detector. Thedetector 11212 is configured to detect an optical signal, e.g.,luminescence, produced due to a chemical reaction in a container, e.g.,container assembly 3700 as described herein. In some embodiments, thedetector 11212 can include an amperometric detector, potentiometricdetector, conductometric, and/or impedometric detector, configured todetect a current, voltage, or conductance, resistance and/or impedancechange produced by the reporter, e.g., luciferase. A bottom surface ofthe detector enclosure 11214 is coupled to a mount 11216 that isdisposed on the base plate 11211. The mount 11216 can be made from arigid but soft material, e.g., rubber or foam pad, to provide acushioned support to the detector enclosure 11214. A top surface of thedetector enclosure 11214 is coupled to a separator 11218, e.g., screwed,bolted, and/or riveted to the separator 11218. The separator 11218 canbe made from strong, rigid and low friction material, e.g., a polishedmetal plate (e.g., aluminum, stainless steel, etc.). The separator 11218is configured to provide a separation layer between the detectorenclosure 11214 and the shutter 11230, e.g., to prevent wear of thedetector enclosure 11214 due to a displacement of the shutter 11230, asdescribed herein. The separator 11218 includes an aperture 11219 whichis configured such that when the separator 11218 is coupled to thedetector 11212, the aperture 11219 is located directly above thedetector 11212 and provides unhindered optical access to the detector11212. The aperture 11219 also includes a window 11220, e.g., a circularglass or transparent plastic piece, disposed therein. The window 11220is configured to protect the detector 11212, e.g., from dust particlesand/or physcial damage due to accidental contact by an end of acontainer, e.g., container assembly 3700. A seat 11222 is also disposedin the aperture 11219, configured to securely seat the window 11220 inthe aperture 11219. The seat 11222 can also be configured to contact atop surface of the detector enclosure 11214 and encircling the detector11212, for example, to light seal the detector 11212 from ambient light.In some embodiments, the seal 11214 can be formed from rubber, plasticand/or polymers and can be an o-ring. A seal 11224, e.g., a rubber seal,is also disposed on the separator 11224. The seal 11224 can beconfigured to light seal the internal volume 11210 of the housing 11202,e.g., the internal volume 11210 housing the shutter 11230 from ambientlight, e.g., to reduce background noise, increase signal quality and/orrepeatability.

The detector assembly 11200 also includes a shutter 11230 slidablydisposed on the separator 11218 and configured to move from a firstshutter position wherein the shutter 11230 is closed and the detector11212 is optically uncoupled with a container, e.g., container assembly3700 disposed in the detector assembly 11200, to a second shutterposition wherein the shutter 11230 is open and the detector 11212 isoptically coupled to the container. As shown in FIG. 89-90, the shutter11230 includes a recess 11232, which includes a slot 11234 and a surface11236. The slot 11234 is shaped and sized to receive an end portion of acontainer, e.g., container assembly 3700. The surface 11236 iscontoured, e.g., curved to conform to a contour of a bottom end of thecontainer. The surface 11236 is inclined at angle leading from a topsurface 11236 of the shutter 11230 to a top edge of the slot 11234. Insome embodiments, the surface 11236 can be inclined at an angle of 30degrees, 40 degrees, 45 degrees, 50 degrees, or 60 degrees from a tophorizontal surface of the shutter 11230. The surface 11230 is configuredto be engaged by a bottom end of the container, e.g., container assembly3700, to manipulate the shutter 11230 from the first position to thesecond position, as described herein. The shutter 11230 includes asleeve 11238 having a spring 11240 disposed therein. A second end of thespring 11240 is disposed in a notch 11241 in the housing 11202 (FIG. 85)mounted on a pin 11242. The spring 11240 is configured to urge theshutter 11230 from the second shutter position to the first shutterposition, and/or secure, hold and/or prevent lateral movement of acontainer, e.g., container assembly 3700 disposed in the slot 11234, asdescribed herein.

The shutter 11230 further includes a channel 11244 configured to definean optical pathway for an electromagnetic radiation, for example,luminescence emitted by a light source 11246. The channel 11244 isconfigured such that the channel 11244 optically couples the lightsource 11246 to the detector 11212 only when the shutter is in the firstposition. In some embodiments, the light source 11246 can include alight emitting diode (LED) or a laser, and can further include a lightguide, e.g., a fiber optic cable. The light source 11246 can beconfigured to emit a reference luminescent signal, e.g., a calibratingsignal for calibrating the detector 11212.

As described herein, the shutter 11230 is configured to displace from afirst position within the internal volume 11210, wherein the shutter11230 is closed, to a second position wherein the shutter 11230 is open.FIG. 91-94 show a side cross-section of the detector assembly 11200 in afirst configuration, a second configuration, a third configuration and afourth configuration. In the first configuration (FIG. 72), no containeris disposed in the detector assembly 11200. The spring 11240 applies aforce on the shutter 11230 along a horizontal axis H_(A) of the shutterin a direction shown by the arrow AA to manipulate and maintain theshutter 11230 into the first shutter position, such that the slot 11234of the shutter 11230 is not aligned with the aperture 11219 and thedetector 11212, such that no ambient light is incident on the detector11212. The channel 11244 is aligned with the detector 11212, such thatthe light source 11246 is optically coupled to the detector 11212.Therefore, in the first configuration, the light source 11246 can beused to send a reference signal to the detector 11212, e.g., tocalibrate the detector 11212.

In the second configuration (FIG. 92), a container assembly 3700 asdescribed before herein, is disposed in the detector assembly 11200 in afirst position. The container assembly 3700 includes a reaction chamber3732 and a reagent module 3740 as described before herein. The containerassembly 3700 can be disposed in the detector assembly 11200, e.g., bythe manipulator assembly 11600. The reaction chamber 3732 of thecontainer assembly 3700 is substantially disposed in the channel 11209while at least a portion of the reagent module 3740 is disposed in thereceptacle 11204. An end portion 3733 of the reaction chamber 3732 is incontact with the surface 11236. A downwards force is applied on thecontainer assembly 3700, e.g., by the manipulator assembly 11600 asdescribed herein, along a vertical axis VA of the detector assembly11200 as shown by the arrow F which is communicated to the surface 11236of the shutter 11230 by the end portion 3733 of the reaction chamber3732 included in container assembly 3700. Because the surface 11236 isinclined, e.g., at 45 degrees with respect to a horizontal axis of theshutter 11230, the end portion 3733 of the container assembly 3700exerts an angular force Fxy on the surface 11236 as shown. The force Fxyhas a horizontal component Fx and a vertical component Fy as shown inFIG. 92. The horizontal component Fx urges the shutter 11230 assembly todisplace horizontally along the horizontal axis H_(A) in the directionindicated by the arrow Fx such that the slot 11234 of the shutter 11230displaces towards the detector 11212 as shown in the third configuration(FIG. 93). The spring 11240 can be configured to exert a reactive forceon reaction chamber 3732, but not large enough to prevent the reactionchamber 3732 from manipulating the shutter 11230. The channel 11209 ofthe housing 11202 can be in close tolerance or only slightly larger thanthe diameter of the reaction chamber 3732, e.g., to prevent any lateralmovement of the container assembly 3700 by the reactive force exerted bythe spring 11240.

The downward force F on the container assembly 3700 can be maintaineduntil in the fourth configuration, the shutter 11230 is displaced to asecond position such that the slot 11234 is aligned with the detector11232 and the end portion 3733 of the reaction chamber 3732 is disposedin the slot 11234, as shown in FIG. 94. The end portion 3733 of thereaction chamber 3732 can be proximal to but not contacting the window11220, e.g., to prevent scratching and/or wear of the window. In thisconfiguration, the spring 11240 applies a force on the shutter 11230urging the shutter 11230 towards the first shutter position. This forceis communicated to a side wall of the end portion 3733 of the reactionchamber 3732 through a side wall of the slot 11234 included in theshutter 11230, which prevents the shutter 11230 from sliding into thefirst shutter position. In some embodiments, the slot 11234 can define adetection volume configured to restrict any signal produced in thereaction chamber 3732, e.g., luminescence produced by the interaction ofa reporter molecule, e.g., luciferase, with a substrate, e.g.,tridecanal, for example, to increase signal quality, sensitivity,repeatability and/or reduce background noise. Furthermore, a bottomsurface 3735 of the reagent module 3740 included in the containerassembly 3700 rests on and is flush with the gasket 11206 such that noambient light can enter the housing 11202 of the detector assembly11200. In some embodiments, the manipulator assembly 11600 can maintainthe downward force F on the container assembly 3700 in the fourthconfiguration, e.g., to maintain strong contact between the bottomsurface 3735 of the reagent module 3740 included in the containerassembly 3700, and the gasket 11206

FIG. 95 shows the detector circuitry 11270 that can be used to controldetector assembly 11200, according to an embodiment. The circuitry 11270is disposed in the housing 11202 of the instrument 11200, mounted on thebase plate 11118. In some embodiments, the circuitry 11270 can include aphoton detector and/or any other circuitry for processingopto-electronic signals, as are commonly known in the arts.

In some embodiments, a reagent, e.g., a substrate can be communicatedinto the reaction chamber 3732 in the fourth configuration such that achemical reaction produces a signal in the reaction chamber 3732 whichcan be detected by the detector 11212. For example, the second plunger11636 included in the manipulator assembly 11600 can engage the actuator3760 included in the reagent module 3740 of the container assembly 3700to communicate a substrate, e.g., tridecanal or any other substratedescribed herein, into the reaction chamber 3732. The substrate caninteract with a reporter molecule present in a sample solution disposedin the container, e.g., the reporter molecule luciferase or any otherreporter molecule described herein produced by the interaction of abiologic or abiologic vector such as a transduction particle (e.g.,transduction particle 160 or any other transduction particle describedherein) with a target cell, e.g., bacteria such as MRSA. The interactionof the substrate and reporter molecule can produce a signal, e.g.,luminescence that is detected by the detector 11212. In someembodiments, the reaction between the substrate and the reportermolecule can be an instantaneous, e.g., flash reaction, such that asignal is produced instantly after communication of the substrate intothe sample solution including the reporter molecules. Detection of thesignal indicates that the sample disposed in the reaction chamber 3732contains the target cell.

As described herein, the detector 11212 or any other detector describedherein can be calibrated before making a signal measurement. FIG. 96illustrates a flow diagram of a method for calibrating and making ameasurement with a detector, e.g., detector 11212 included in aninstrument, e.g., instrument 11000. The method includes receiving afirst signal associated with a magnitude of light emission in adetection volume, 702, at a first time such that the detection volume isoptically isolated from a channel by a movable shutter, e.g., shutter11230, which is disposed in a first shutter position. In someembodiments, a light emission e.g., a calibration signal, can betransmitted into the detection volume via a light channel defined by theshutter when the shutter is in the first position. A force is applied tosample container to move the shutter into the second position, 704. Thecontainer can initially be in the at least partially disposed within thechannel such that the application of a force on the container, allows adistal end portion of the container to move the shutter from the firstshutter position to the second shutter position. This allows the distalend portion of the container to move into the detection volume, 706,such that the channel is now in optical communication with the detectionvolume. In this position a second signal associated with a lightemission in the detection volume can be received, 708. In someembodiments, before receiving the second signal, a reagent, e.g., asubstrate can be conveyed into the distal end portion of the container.The substrate, e.g., tridecanal can be configured to react with, forexample, reporter molecules present in the sample to produce a lightemission. In some embodiments, the detector 11212, or any other detectordescribed herein, can include internal calibration controls, e.g.,software algorithms, such that an external calibration light source isnot required.

As described herein, the instrument 11000 or any other instrumentdescribed herein can be used to manipulate a container (e.g., containerassembly 3700 or any other container described herein), for example, totransport a container, communicate reagents into a reaction volume ofthe container and/or detect a signal, e.g., luminescence produced withinthe container. FIG. 97 illustrates a flow chart of a method formanipulating a container within an instrument. A container that can havea sample disposed therein containing target cells, e.g., bacteria, canbe loaded into a loading zone of the instrument 802, e.g., the loadingcartridge 11300. The container is transported to a heater included inthe instrument 804, e.g., by the manipulator assembly 11600 via thedrive assembly 11500 to the heater assembly 11400. A biologic orabiologic vector such as a transduction particle is communicated into areaction volume of the container 806, e.g., by a manipulation of theinner plunger 11632 of the manipulator assembly 11600. The container ismaintained at a predetermined temperature for a predetermined time 808,e.g., at 37 degrees Celsius for 4 hours by the heater assembly 11400.The transduction particles interact with the target cells included inthe sample, such that the target cells produce a series of reportermolecule 810. In some embodiments, the heater assembly 11400 can beconfigured maintain the container (e.g., container assembly 3700 or anyother container described herein) at a series of temperatures, forpredetermined times. For example, the container and the sample disposedtherein can be maintained at 37 degrees Celsius for a first time, e.g.,4 hours. The temperature of the container can then be lowered to asecond temperature lower than the first temperature (e.g., 30 degreesCelsius), for example, by transferring to a heating block 11422 at thesecond temperature. The container can, for example, be maintained at thesecond temperature until detection. The container is then transported toa detector included in the instrument 812, e.g., the detector assembly11200. For example, the manipulator assembly 11600 can be used totransport the container via the drive assembly 11500. A substrate isthen communicated into the container 814, e.g., via a manipulation ofthe outer plunger 11636 of the manipulator assembly 11600. The substrateinteracts with reporter molecules to produce a signal 816 that isdetected using the detector 818. The analyzed container is thentransported to an unloading zone of the instrument 820, e.g., anunloading cartridge 11300, and can be removed from the container 822.

In some embodiments, an instrument, e.g., instrument 11000 or any otherinstrument described herein, can be in communication with a laboratoryinformation system (LIS), e.g., the LIS 1900 as shown in FIGS. 2-3.

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Where methods and/or schematics described above indicatecertain events and/or flow patterns occurring in certain order, theordering of certain events and/or flow patterns may be modified.Additionally, certain events may be performed concurrently in parallelprocesses when possible, as well as performed sequentially. While theembodiments have been particularly shown and described, it will beunderstood that various changes in form and details may be made.

In some embodiments, the fluidic pathways defined by any of the reagentmodules (e.g., 1740) can include valves or any other flow controlmechanism. Such mechanisms include a flap valve, membrane valve,duckbill valve, umbrella valve, septum, or any other suitable valvingmechanism to allow reagents to flow in one direction. In someembodiments, the valves can be pressure sensitive such that they allowfluid communication only above a predefined pressure threshold, e.g., toprevent accidental communication of reagents.

In some embodiments, any of the systems and methods described hereininclude a reporter system that reports on the presence of inducermolecules within target cells can be developed by incorporating into anon-replicative transduction particle, of the types shown and describedherein, a reporter gene that is operably linked to an inducible promoterthat controls the expression of a target gene within a target cell. Whenthe reporter vector is introduced into the target cell that expressesthe inducer of the target gene promoter, expression of the reporter geneis possible via induction of the target gene promoter in the reportervector.

In one embodiment, a VanR reporter system can be developed for thepurpose of detecting Vancomycin Resistant Enterococci (VRE). The Tn1546transposon that can be present in E. faecium may contain the vanRinducer gene and the vanA target gene. A VRE reporter system can bedeveloped by developing an E. faecium-targetting non-replicativetransduction particle that incorporates a reporter gene operativelylinked to the promoter PH that controls the expression of the vanHAXoperon that includes the vanA gene. When the transduction particledelivers the P_(H)-controlled reporter gene into the E. faecium cell,the reporter gene will be expressed via induction of the PH promoter bythe product of vanR.

In another embodiment, a reporter system for detecting TcdD, the inducerof the promoters of the toxins A and B genes (tcdA and tcdB,respectively) of C. difficile can be developed by developing anon-replicative transduction particle targeting C. difficile and thatincorporates reporter gene that is operatively linked to the tcdA genepromoter. The PaLoc transposon in C. difficile may contain the tcdD geneand the tcdA target gene. In the native cell, when the tcdD gene isexpressed and produces the TcdD protein, TcdD is able to induce PtcdA inthe PaLoc transposon thus causing the expression of the tcdA gene andthus producing the toxin A protein. By introducing a reporter gene thatoperatively linked to the tcdA gene promoter (PtcdA) into a target cell,TcdD is then also able to induce PtcdA that controls the reporter gene,thus causing the expression of a reporter molecule.

A reporter system can be developed for reporting on the presence of atarget intracellular enzyme by developing a non-replicative transductionparticle-based viable cell reporter that employs a reporter thatrequires a substrate for signal generation and by caging the substratesuch that it is not capable of triggering a signal via the reporterunless the substrate is un-caged via interacting with the target enzyme.A target cell is exposed to the transduction particle such that thereporter is expressed within the target cell and the caged substrate isapplied. If the target cell contains a target enzyme, an interactionbetween the target enzyme and the caged substrate un-cages the substratethus allowing the un-caged substrate to trigger a signal from theexpressed reporter molecules.

In one embodiment, the reporter molecule to be expressed can be Renillaluciferase and the caged substrate can be Renilla luciferin that iscaged such that a β-lactamase enzyme that is endogenous to the targetcell is able to cleave the caging compound from the caged luciferin andrelease un-caged luciferin. By incorporating these components into anon-replicative transduction particle that targets a cell that maycontain a β-lactamase enzyme, a target-cell-specific β-lactamase enzymereporter system can be developed.

An intracellular molecule reporter system can be developed byincorporating into a non-replicative transduction particle a switchablereporter molecule that does not emit a signal unless it interacts withan intracellular target molecule.

In one embodiment, a non-replicative transduction particle can bedesigned to incorporate a gene expressing switchable aptamer designed toundergo a conformational change upon its binding to an intracellulartarget molecule. The conformational change allows the aptamer to thenbind a fluorophore that exhibits enhanced fluorescence when bound by theaptamer.

An antisense RNA-based reporter system for detecting target transcriptswithin viable cells by causing the expression of a reporter molecule ifa target transcript is present within a cell can be developed. In thegeneral embodiment a non-replicative transduction particle incorporatesa DNA sequence encoding an antisense message that is complementary to aregion of a target transcript (target), and a sequence encoding amutated version of the target transcript (target*) fused to a reportergene (reporter) is used. The mutation of the target transcript is suchthat the antisense transcript binds to the mutated target transcriptwith a lower affinity than that of it's binding to the native targettranscript. The antisense sequence is controlled by a promoter sequence(P), and the mutated target sequence linked to the reporter gene iscontrolled by an identical promoter sequence (P). When the reportersystem is introduced into a cell that does not contain an endogenoustarget transcript, the expressed antisense transcript inhibits thetranslation of the reporter gene and the antisense transcript andreporter transcript are consumed in the process. However, when thevector is inserted into a cell that does contain an endogenous targettranscript, the expressed antisense transcript preferably binds to thenative target transcript and the antisense transcript and targettranscript are consumed in the process, leaving the reporter gene to betranslated thus producing a reporter protein that may be detected. Inthis manner, this vector causes the expression of a detectable signalwhen it is introduced into a target cell containing the targettranscript.

In some embodiments, non-replicative transduction particles targeting S.aureus cells are designed to report on the presence of mecA transcriptsthus resulting in a MRSA detection system. The transduction particlesdeliver DNA sequences that encode an antisense message that iscomplementary to a region of mecA transcript (mecA), and a sequenceencoding a mutated version of mecA transcript (mecA*) fused to thebacterial luciferase genes luxA and luxB (luxAB). The mutation of themecA* transcript is such that the antisense transcript binds to itstranscript with a lower affinity than that of it's binding to the nativemecA transcript. The mecA*-luxAB fusion and the antisense mecA genefragment are each operatively linked to the constitutively expressedpromoters. When the reporter construct is introduced into MRSA cells bythe transduction particle, if the cell does not produce an endogenousmecA transcript, then the antisense sequence fragment of the mecA genecan only bind to the mecA sequence of the transcript of the modifiedfragment of the mecA gene fused to the luxAB genes. This binding eventthen prevents the translation of the luxAB genes thus preventing theproduction of luciferase within this cell. If, on the other hand, celldoes contain an endogenous mecA transcript, then the transcript of theantisense sequence fragment of the mecA gene will preferentially bind tothe endogenous mecA transcript over the transcript of the modifiedfragment of the mecA gene fused to the luxAB genes thus leaving thistranscript available for translation of the luxAB genes therebyproducing luciferase. In this manner, the mecA transcript reportervector can report on the presence of endogenous mecA transcripts withina cell.

Although various embodiments have been described as having particularfeatures and/or combinations of components, other embodiments arepossible having a combination of any features and/or components from anyof embodiments as discussed above.

What is claimed is:
 1. A method of detecting the presence of a targetcell within a biological sample using an instrument, comprising: mixinga plurality of transduction particles associated with the target cellwith the biological sample, the plurality of transduction particlesengineered to be non-replicative and to include a nucleic acid moleculeformulated to cause the target cell to produce a plurality of reportermolecules, the plurality of transduction particles formulated to bind toand deliver the nucleic acid molecule into the target cell; maintaining,after the mixing, the biological sample and the plurality oftransduction particles for a time period to allow the plurality ofreporter molecules to be produced within the biological sample when thetarget cell is present in the biological sample; placing a reactionchamber containing the biological sample in optical communication with adetector of the instrument; conveying, via a delivery member, a reagentinto the reaction chamber, the reagent formulated to react with theplurality of reporter molecules to catalyze production of a signalassociated with a quantity of the plurality of the reporter molecules,the reagent being conveyed via the delivery member at an angle and in adirection nonperpendicular to a surface of the biological sample withinthe reaction chamber; and receiving, via the detector of the instrument,the signal associated with the quantity of the plurality of reportermolecules.
 2. The method of claim 1, wherein the angle at which thereagent is conveyed is between 15 and 45 degrees relative to alongitudinal axis of the reaction chamber.
 3. The method of claim 1,wherein the conveying the reagent is performed such that the reagent isfurther conveyed with a swirling motion into the reaction chamber. 4.The method of claim 1, wherein the conveying includes conveying thereagent at a flow rate of at least one milliliter per second.
 5. Themethod of claim 1, wherein the conveying includes moving a plunger in aplunger direction within a reagent volume, a first end portion of thedelivery member disposed within the reagent volume, a second end portionof the delivery member disposed within the reaction chamber, a flow ofthe reagent exiting the second end portion of the delivery member in anexit direction nonparallel to the plunger direction.
 6. The method ofclaim 1, further comprising: maintaining a position of the reactionchamber relative to the detector between the conveying and thereceiving.
 7. The method of claim 6, wherein the maintaining theposition includes limiting at least one of lateral or vertical motion ofthe reaction chamber.
 8. The method of claim 1, further comprising:maintaining a distance between reaction chamber and the detector of theinstrument during the receiving.
 9. The method of claim 1, wherein: thereporter molecule is any one of a bacterial luciferase, an eukaryoticluciferase, a fluorescent protein, an enzyme suitable for colorimetricdetection, a protein suitable for immunodetection, a peptide suitablefor immunodetection or a nucleic acid that function as an apatamer orthat exhibits enzymatic activity; and the reagent includes tridecanal.10. The method of claim 1, wherein the receiving is performed for lessthan sixty seconds after the conveying.
 11. The method of claim 1,wherein: the sample is a raw sample that has not undergone a separationoperation or a washing operation; and the target cell within the sampleis non-isolated.
 12. The method of claim 1, wherein: the signal is alight emission associated with a flash luminescence reaction; and thereagent comprises a fatty aldehyde formulated to catalyze theluminescence reaction.
 13. The method of claim 1, wherein: the reactionchamber includes a flat base; and the placing includes putting thebiological sample in optical communication with the detector of theinstrument via the flat base.
 14. A method of detecting the presence ofa target cell within a biological sample using an instrument,comprising: placing a reaction chamber containing a sample in opticalcommunication with a detector of the instrument, the biological samplecontaining a plurality of reporter molecules; conveying, via a deliverymember, a reagent into the reaction chamber at an angle relative to alongitudinal axis of the reaction chamber, the reagent formulated toreact with the plurality of reporter molecules to enhance the productionof a signal associated with a quantity of the plurality of the reportermolecules; and receiving, via the detector of the instrument, the signalassociated with the quantity of the plurality of reporter molecules. 15.The method of claim 14, wherein the conveying the reagent is performedsuch that the reagent is further conveyed with a swirling motion intothe reaction chamber.
 16. The method of claim 14, wherein the conveyingincludes conveying the reagent at a flow rate of at least one milliliterper second.
 17. The method of claim 14, wherein the delivery member ispositioned such that the conveying the reagent produces acircumferential flow of the reagent.
 18. The method of claim 14, furthercomprising maintaining a distance between reaction chamber and thedetector of the instrument during the receiving.
 19. The method of claim14, wherein: the signal is a light emission associated with a flashluminescence reaction; and the reagent comprises a fatty aldehydeformulated to catalyze the luminescence reaction.
 20. The method ofclaim 14, wherein: the reaction chamber includes a flat base; and theplacing includes putting the biological sample in optical communicationwith the detector of the instrument via the flat base.